Modified amylases from pseudomonas species, methods of making and uses thereof

ABSTRACT

Variant polypeptides derivable from a parent polypeptide having non-maltogenic exoamylase activity, in which the variant polypeptides comprise an amino acid mutation at one or more positions selected from the group consisting of: 121, 161, 223, 146, 157, 158, 198, 229, 303, 306, 309, 316, 353, 26, 70, 145, 188, 272, 339, with reference to the position numbering of a  Pseudomonas saccharophila  exoamylase sequence shown as SEQ ID NO: 1.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is the national phase of International PatentApplication PCT/GB2005/002675 filed Jul. 7, 2005 and published as WO2006/003461 on Jan. 12, 2006, which claims priority to U.S. applicationSer. No. 10/886,504, filed Jul. 7, 2004, abandoned, U.S. applicationSer. No. 10/886,505, filed Jul. 7, 2004, abandoned, U.S. applicationSer. No. 10/886,527, filed Jul. 7, 2004, abandoned and U.S. applicationSer. No. 10/947,612, filed Sep. 22, 2004, abandoned and which claimsbenefit of U.S. Application No. 60/608,919, filed Jul. 7, 2004 and whichclaims benefit of U.S. Application No. 60/612,407, filed Sep. 22, 2004.

Reference is made to U.S. provisional applications Ser. Nos. 60/485,413,601485,539 and 60/485,616 filed Jul. 7, 2003. Reference is also made tointernational applications PCT/US2004/021723 and PCI/US2004/021739 filedJul. 7, 2004 and designating the US (applicant: Genencor International,Inc). Reference is also made to U.S. utility application Ser. Nos.10/886,905 and 10/866,903 all of which were also filed Jul. 7, 2004.

Reference is also made to U.S. provisional application Ser. No.60/608,919 (filed as U.S. utility application Ser. No. 10/887,056 onJul. 7, 2004 but converted to a provisional application on Sep. 15,2004). Reference is also made to U.S. provisional application Ser. No.60/612,407 which was filed Sep. 22, 2004.

Reference is additionally made to U.S. application Ser. No. 60/485,539filed Jul. 7, 2003. Reference is also made to international applicationPCT/IB2004/002487 filed Jul. 7, 2004 and designating the US (applicant:Danisco A/S). Reference is also made to U.S. utility application Ser.No. 10/886,023 filed Jul. 7, 2004.

Reference is also made to U.S. utility application Ser. Nos. 10/886,505,10/886,527 and 10/886,504, all of which were filed Jul. 7, 2004.Reference is also made to U.S. utility application Ser. No. 10/947,612filed Sep. 22^(nd), 2004.

The foregoing applications, and each document cited or referenced ineach of the present and foregoing applications, including during theprosecution of each of the foregoing applications (“application andarticle cited documents”), and any manufacturer's instructions orcatalogues for any products cited or mentioned in each of the foregoingapplications and articles and in any of the application and articlecited documents, are hereby incorporated herein by reference.Furthermore, all documents cited in this text, and all documents citedor reference in documents cited in this text, and any manufacturer'sinstructions or catalogues for any products cited or mentioned in thistext or in any document hereby incorporated into this text, are herebyincorporated herein by reference. Documents incorporated by referenceinto this text or any teachings therein may be used in the practice ofthis invention. Documents incorporated by reference into this text arenot admitted to be prior art.

FIELD

This invention relates to polypeptides, specifically amylasepolypeptides and nucleic acids encoding these, and their uses asnon-maltogenic exoamylases in producing food products. The amylases ofthe present invention have been engineered to have more beneficialqualities. Specifically, the amylases of the current invention show analtered exospecifity and/or altered thermostability. In particular, thepolypeptides are derived from polypeptides having non-maltogenicexoamylase activity, in particular, glucan 1,4-alpha-maltotetrahydrolase(EC 3.2.1.60) activity.

BACKGROUND

Improved amylases can ameliorate problems inherent in certain processes,such as baking. Crystallisation of amylopectin takes place in starchgranules days after baking, which leads to increased firmness of breadand causes bread staling. When bread stales, bread loses crumb softnessand crumb moisture. As a result, crumbs become less elastic, and breaddevelops a leathery crust.

Enzymatic hydrolysis (by amylases, for example) of amylopectin sidechains can reduce crystallization and increase anti-staling.Crystallization depends upon the length of amylopectin side chains: thelonger the side chains, the greater the crystallization. Most starchgranules are composed of a mixture of two polymers: amylopectin andamylose, of which about 75% is amylopectin. Amylopectin is a very large,branched molecule consisting of chains of α-D-glucopyranosyl unitsjoined by (1-4) linkages, where the chains are attached by α-D-(1-6)linkages to form branches. Amylose is a linear chain of (1-4) linkedα-D-glucopyranosyl units having few α-D-(1-6) branches.

Baking of farinaceous bread products such as white bread, bread madefrom bolted rye flour and wheat flour and rolls is accomplished bybaking the bread dough at oven temperatures in the range of from 180 to250° C. for about 15 to 60 minutes. During the baking process a steeptemperature gradient (200→120° C.) prevails over the outer dough layerswhere the crust of the baked product is developed. However, due tosteam, the temperature in the crumb is only about 100° C. at the end ofthe baking process. Above temperatures of about 85° C., enzymeinactivation can take place and the enzyme will have no anti-stalingproperties. Only thermostable amylases, thus, are able to modify starchefficiently during baking.

Endoamylase activity can negatively affect the quality of the finalbread product by producing a sticky or gummy crumb due to theaccumulation of branched dextrins. Exo-amylase activity is preferred,because it accomplishes the desired modification of starch that leads toretardation of staling, with fewer of the negative effects associatedwith endo-amylase activity. Reduction of endoamylase activity can leadto greater exospecifity, which can reduce branched dextrins and producea higher quality bread.

SUMMARY

We provide, according to the invention, a PS4 variant polypeptide as setout in the claims. We further provide for the use of such a PS4 variantpolypeptide, including in and as food additives, food products, bakeryproducts, improver compositions, feed products including animal feeds,etc as set out in the claims. We provide for nucleic acids which encodeand which relate to PS4 variant polypeptides, as set out in the claims.Methods for producing such PS4 variant polypeptides, as well as otheraspects of the invention, are also set out in the claims.

SEQUENCE LISTINGS

SEQ ID NO: 1 shows a PS4 reference sequence, derived from Pseudomonassaccharophila maltotetrahydrolase amino acid sequence. SEQ ID NO: 2shows a PSac-D34 sequence; Pseudomonas saccharophila maltotetrahydrolaseamino acid sequence with 11 substitutions and deletion of the starchbinding domain. SEQ ID NO: 3 shows a PSacD20 sequence; Pseudomonassaccharophila maltotetrahydrolase amino acid sequence with 13substitutions and deletion of the starch binding domain SEQ ID NO: 4shows a PSac-D14 sequence; Pseudomonas saccharophila maltotetrahydrolaseamino acid sequence with 14 substitutions and deletion of the starchbinding domain. SEQ ID NO: 5 shows a Pseudomonas saccharophila Glucan1,4-alpha-maltotetrahydrolase precursor (EC 3.2.1.60) (G4-amylase)(Maltotetraose-forming amylase) (Exo maltotetraohydrolase)(Maltotetraose-forming exo-amylase). SWISS-PROT accession number P22963.SEQ ID NO: 6 shows a P. saccharophila mta gene encodingmaltotetraohydrolase (EC number=3.2.1.60). GenBank accession numberX16732. SEQ ID NO:7 shows a PS4 reference sequence, derived fromPseudomonas stutzeri maltotetrahydrolase amino acid sequence. SEQ ID NO:8 shows a PStu-D34 sequence; Pseudomonas stutzeri maltotetrahydrolaseamino acid sequence with 9 substitutions. SEQ ID NO: 9 shows a PStu-D20sequence; Pseudomonas stutzeri maltotetrahydrolase amino acid sequencewith 11 substitutions. SEQ ID NO: 10 shows a PStu-D14 sequence;Pseudomonas stutzeri maltotetrahydrolase amino acid sequence with 12substitutions. SEQ ID NO: 11 shows a Pseudomonas stutzeri (Pseudomonasperfectomarina). Glucan 1,4-alpha-maltotetrahydrolase precursor (EC3.2.1.60) (G4-amylase) (Maltotetraose-forming amylase)(Exo-maltotetraohydrolase)(Maltotetraose-forming exo-amylase).SWISS-PROT accession number P13507. SEQ ID NO: 12 shows a P. stutzerimaltotetraose-forming amylase (amyP) gene, complete cds. GenBankaccession number M24516. SEQ ID NO: 13 shows a pMD55 sequence;Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with11 substitutions (G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N,L178F, A179T and G121F). SEQ ID NO: 13 shows a pMD55 sequence;Pseudomonas saccharophila maltotetrahydrolase amino acid sequence with11 substitutions (G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N,L178F, A179T and G121F) and deletion of the starch binding domain. SEQID NO: 13 shows a pMD55 sequence; Pseudomonas saccharophilamaltotetrahydrolase amino acid sequence with 11 substitutions (G134R,A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F, A179T and G121F).SEQ ID NO: 13 shows a pMD55 sequence; Pseudomonas saccharophilamaltotetrahydrolase amino acid sequence with 11 substitutions (G134R,A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F, A179T and G121F)and deletion of the starch binding domain. SEQ ID NO: 14 shows a PMD96sequence: Pseudomonas saccharophila maltotetrahydrolase amino acidsequence having mutations at N33Y, D34N, G121F, G134R, A141P, I157L,S161A, L178F, A179T, G223E, H307L and S334P. SEQ ID NO: 15 shows a SSM381 sequence: Pseudomonas saccharophila maltotetrahydrolase amino acidsequence having mutations at 33Y, 34N, 121F, 134R, 141P, 146G, 157L,161A, 178F, 179T, 223E, 307L and 334P. SEQ ID NO: 16 shows a SSM279 B1sequence: Pseudomonas saccharophila maltotetrahydrolase amino acidsequence having amino acid mutations at 33Y, 34N, 121F, 134R, 141P,157M, 161A, 178F, 179T, 223E, 307L and 334P. SEQ ID NO: 17 shows aSSM237 P2 sequence: Pseudomonas saccharophila maltotetrahydrolase aminoacid sequence having amino acid mutations at 33Y, 34N, 121F, 134R, 141P,157L, 158T, 161A, 178F, 179T, 223E, 307L and 334P. SEQ ID NO: 18 shows aPseudomonas saccharophila maltotetrahydrolase amino acid sequence havingamino acid mutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F,179T, 198W, 223E, 307L and 334P. SEQ ID NO: 19 shows a SSM325 F3sequence: Pseudomonas saccharophila maltotetrahydrolase amino acidsequence having mutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A,178F, 179T, 223E, 229P, 307L and 334P. SEQ ID NO: 20 shows a pMD129sequence: Pseudomonas saccharophila maltotetrahydrolase amino acidsequence having mutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A,178F, 179T, 198W, 223E, 229P, 307L and 334P. SEQ ID NO: 21 shows aSSM341 A9 sequence: Pseudomonas saccharophila maltotetrahydrolase aminoacid sequence having mutations at 33Y, 34N, 121F, 134R, 141P, 157L,161A, 178F, 179T, 223E, 303E, 307L and 334P. SEQ ID NO: 22 shows aSSM341 G11 sequence: Pseudomonas saccharophila maltotetrahydrolase aminoacid sequence having mutations at 33Y, 34N, 121F, 134R, 141P, 157L,161A, 178F, 179T, 223E, 303D, 307L and 334P. SEQ ID NO: 23 shows aSSM350 B11 sequence: Pseudomonas saccharophila maltotetrahydrolase aminoacid sequence having mutations at 33Y, 34N, 121F, 134R, 141P, 1571,161A, 178F, 179T, 223E, 306T, 307L and 334P. SEQ ID NO: 24 shows aSSM350 C12 sequence: Pseudomonas saccharophila maltotetrahydrolase aminoacid sequence having mutations at 33Y, 34N, 121F, 134R, 141P, 157L,161A, 178F, 179T, 223E, 306G, 307L and 334P. SEQ ID NO: 25 shows aSSM332 Q4 sequence: Pseudomonas saccharophila maltotetrahydrolase aminoacid sequence having amino acid mutations at 33Y, 34N, 121F, 134R, 141P,157L, 161A, 178F, 179T, 223E, 309P, 307L and 334P. SEQ ID NO: 26 shows aSSM365 B4 sequence: Pseudomonas saccharophila maltotetrahydrolase aminoacid sequence having amino acid mutations at 33Y, 34N, 121F, 134R, 141P,157L, 161A, 178F, 179T, 223E, 307L, 316S, and 334P. SEQ ID NO: 27 showsa SSM365 F4: Pseudomonas saccharophila maltotetrahydrolase amino acidsequence having mutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A,178F, 179T, 223E, 307L, 316P and 334P. SEQ ID NO: 28 shows a SSM360 C7:Pseudomonas saccharophila maltotetrahydrolase amino acid sequence havingmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,307L, 334P and 353T.

DETAILED DESCRIPTION

In the following description and examples, unless the context dictatesotherwise, dosages of PS4 variant polypeptides are given in parts permillion (micrograms per gram) of flour. For example, “1 D34” as used inTable 2 indicates 1 part per million of pSac-D34 based on weight perweight. Preferably, enzyme quantities or amounts are determined based onactivity assays as equivalents of pure enzyme protein measured withbovine serum albumin (BSA) as a standard, using the assay described inBradford (1976, A rapid and sensitive method for the quantification ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal. Biochem. 72:248-254).

In describing the different PS4 variant polypeptide variants produced orwhich are contemplated to be encompassed by this document, the followingnomenclature will be adopted for ease of reference:

-   -   (i) where the substitution includes a number and a letter, e.g.,        141P, then this refers to [position according to the numbering        system/substituted amino acid].    -   Accordingly, for example, the substitution of an amino acid to        proline in position 141 is designated as 141P;    -   (ii) where the substitution includes a letter, a number and a        letter, e.g., A141P, then this refers to [original amino        acid/position according to the numbering system/substituted        amino acid]. Accordingly, for example, the substitution of        alanine with proline in position 141 is designated as A141P.

Where two or more possible substituents are possible at a particularposition, this will be designated by contiguous letters, which mayoptionally be separated by slash marks “/”, e.g., G303ED or G303E/D.Where the relevant amino acid at a position can be substituted by anyamino acid, this is designated by [position according to the numberingsystem/X], e.g., 121X.

Multiple mutations may be designated by being separated by slash marks“/”, e.g. A141P/G223A representing mutations in position 141 and 223substituting alanine with proline and glycine with alanine respectively.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with ageneral dictionary of many of the terms used in this invention. Althoughany methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,the preferred methods and materials are described. Numeric ranges areinclusive of the numbers defining the range. Unless otherwise indicated,nucleic acids are written left to right in 5′ to 3′ orientation; aminoacid sequences are written left to right in amino to carboxyorientation, respectively.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-314-2), 1855, Lars-Inge Larsson“Immunocytochemistry: Theory and Practice”, CRC Press inc., Baca Raton,Fla., 1988, ISBN 0-8493-6078-1, John D. Pound (ed); “ImmunochemicalProtocols, vol 80”, in the series: “Methods in Molecular Biology”,Humana Press, Totowa, N.J., 1998, ISBN 0-89603493-3, Handbook of DrugScreening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes(2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref:A Handbook of Recipes, Reagents, and Other Reference Tools for Use atthe Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold SpringHarbor Laboratory, ISBN 0-87969-630-3. Each of these general texts isherein incorporated by reference.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

PS4 Variant Polypeptides

We provide a non-maltogenic exoamylase having a substitution at one ormore positions which effect an altered property, preferably alteredexospecificity or altered thermostability, or both, relative to theparent enzyme.

We fisher provide for compositions, including food additives, foodproducts, bakery products, improver compositions, feed productsincluding animal feeds, etc comprising polypeptides which havenon-maltogenic exoamylase activity, as well as methods of making andusing such polypeptides and the compositions. We provide for other usesof such compositions such as in the preparation of detergents, assweeteners, syrups, etc. The compositions include the polypeptidetogether with at least one other component. In particular, we providefor food or feed additives comprising the polypeptides.

Such polypeptides and nucleic acids vary from their parent sequences byincluding a number of mutations, and are known for convenience as “PS4variant polypeptides”. In other words, the sequence of the PS4 variantpolypeptide or nucleic acid is different from that of its parent at anumber of positions or residues. In preferred embodiments, the mutationscomprise amino acid substitutions, that is, a change of one amino acidresidue for another. Thus, the PS4 variant polypeptides comprise anumber of changes in the nature of the amino acid residue at one or morepositions of the parent sequence.

As used herein, the term “variant” should be taken to mean a moleculebeing derivable from a parent molecule. Variants include polypeptides aswell as nucleic acids. Variants include substitutions, insertions,transversions and inversions, among other things, at one or morelocations. Variants also include truncations. Variants includehomologous and functional derivatives of parent molecules. Variantsinclude sequences that are complementary to sequences that are capableof hybridising to the nucleotide sequences presented herein.

Substitutions for PS4 Variant Polypeptides

We provide for PS4 variant polypeptides with sequence alterationscomprising amino acid substitutions in a non-maltogenic exoamylasesequence. The amino acid substitution may be at any one or more ofpositions 26, 70, 121, 145, 146, 157, 158, 161, 188, 198, 223, 229, 272,303, 306, 309, 316, 339 and 353 with reference to the position numberingof a Pseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO:1.

The amino acid mutation may be at one or more positions selected fromthe groups consisting of: (a) positions 121, 161, 223; (b) positions146, 157, 158, 198, 229, 303, 306, 309, 316, 353; and (c) positions 26,70, 145, 188, 272, 339; and any combination of (a), (b) and (c).

The amino acid substitution may comprise a change to 121F, 121Y, 121W,161A, 223E, 223K. Alternatively, or in addition, the amino acidsubstitution may comprise a change to 146G, 146M, 157MK 158T, 158A,158S, 198W, 198F, 229P, 303E, 303D, 306T, 306G, 309P, 316S, 316P, 316K,316Q, 353T. Further alternatively, or in addition, the amino acidsubstitution may comprise a change to 26E, 70D, 1145D, 188S, 188T, 188H,272Q, 339A, 339E.

Such variant polypeptides are referred to in this document as “PS4variant polypeptides”. Nucleic acids encoding such variant polypeptidesare also disclosed and will be referred to for convenience as “PS4variant nucleic acids”. PS4 variant polypeptides and nucleic acids willbe described in further detail below.

The “parent” sequences, i.e., the sequences on which the PS4 variantpolypeptides and nucleic acids are based, preferably are polypeptideshaving non-maltogenic exoamylase activity. The terms “parent enzymes”and “parent polypeptides” should be interpreted accordingly, and takento mean the enzymes and polypeptides on which the PS4 variantpolypeptides are based. They are described in further detail below.

In particularly preferred embodiments, the parent sequences arenon-maltogenic exoamylase enzymes, preferably bacterial non-maltogenicexoamylase enzymes. In highly preferred embodiments, the parent sequencecomprises a glucan 1,4-alpha-maltotetrahydrolase (EC 3.2.1.60).Preferably, the parent sequence is derivable from Pseudomonas species,for example Pseudomonas saccharophilia or Pseudomonas stutzeri.

In some embodiments, the parent polypeptide comprises, or is homologousto, a wild type non-maltogenic exoamylase sequence, e.g., fromPseudomonas spp.

Thus, the parent polypeptide may comprise a Pseudomonas saccharophilianon-maltogenic exoamylase having a sequence shown as SEQ ID NO: 1. Inother preferred embodiments, the parent polypeptide comprises anon-maltogenic exoamylase from Pseudomonas stutzeri having a sequenceshown as SEQ ID NO: 11, or a Pseudomonas stutzeri non-maltogenicexoamylase having SWISS-PROT accession number P13507.

On the other hand, the parent polypeptide may be a variant of any of thewild type sequences, that is to say, the parent polypeptide may itselfbe engineered, or comprise a PS4 variant polypeptide.

However, it will be clear to the skilled reader that although the PS4variant polypeptides may be derivable by mutating already mutatedsequences, it is possible to construct such variant polypeptides bystarting from a wild type sequence (or indeed any suitable sequence),identifying the differences between the starting sequence and thedesired variant, and introducing the required mutations into thestarting sequence in order to achieve the desired variant.

Proteins and nucleic acids related to, preferably having sequence orfunctional homology with Pseudomonas saccharophilia non-maltogenicexoamylase sequence shown as SEQ ID NO: 1 or a Pseudomonas stutzerinon-maltogenic exoamylase having a sequence shown as SEQ ID NO: 11 arereferred to in this document as members of the “PS4 family”. Examples of“PS4 family” non-maltogenic exoamylase enzymes suitable for use ingenerating the PS4 variant polypeptides and nucleic acids are disclosedin further detail below.

The PS4 variant polypeptides described in this document preferablyretain the features of the parent polypeptides, and additionallypreferably have additional beneficial properties, for example, enhancedactivity or thermostability, or pH resistance, or any combination(preferably all). This is described in further detail below.

The PS4 substitution mutants described here may be used for any suitablepurpose. They may preferably be used for purposes for which the parentenzyme is suitable. In particular, they may be used in any applicationfor which exo-maltotetraohydrolase is used. In highly preferredembodiments, they have the added advantage of higher thermostability, orhigher exoamylase activity or higher pH stability, or any combination.Examples of suitable uses for the PS4 variant polypeptides and nucleicacids include food production, in particular baking, as well asproduction of foodstuffs; further examples are set out in detail below.

The PS4 variant polypeptides may comprise one or more further mutationsin addition to those positions set out above. There may be one, two,three, four, five, six, seven or more mutations preferably substitutionsin addition to those already set out. Other mutations, such asdeletions, insertions, substitutions, transversions, transitions andinversions, at one or more other locations, may also be included. Inaddition, the PS4 variants need not have all the substitutions at thepositions listed. Indeed, they may have one, two, three, four, or fivesubstitutions missing, i.e., the wild type amino acid residue is presentat such positions.

PS4 Variant Polypeptides Based on Wild Type Sequences

In embodiments where the parent polypeptide comprises a wild typesequence, the PS4 variant polypeptides may comprise a wild type sequencebut with a mutation at any one or more of positions 121, 161 and 223,preferably 121F, 121Y, 121W, 161A, 223E or 223K, more preferably G121F,G121Y, G121W, S161A, G223E or G223K.

The PS4 variant polypeptides may comprise a wild type sequence, or asequence as set out in the preceding paragraph, but with a mutation atany one or more of positions 146, 157, 158, 198, 229, 303, 306, 309, 316or 353, preferably 146G, 146M, 157M, 158T, 158A, 158S, 198W, 198F, 229P,303E, 303D, 306T, 306G, 309P, 316S, 316P, 316K, 316Q or 353T, morepreferably 146G, 157M, 158T, 198W, 229P, 303E, 303D, 306T, 306G, 309P,316S, 316P or 353T.

The PS4 variant polypeptides may comprise a wild type sequence, or asequence as set out in the preceding two paragraphs, but with a mutationat any one or more of positions 26, 70, 145, 188, 272 or 339, preferably26E, 26D, 70D, 145D, 188S, 188T, 188H, 272Q, 339A or 339E, morepreferably N26E, N26D, G70D, N145D, G188S, G188T, G188H, H272Q, W339A orW339E.

In general, any combination of the positions, substitutions and specificmutations may be combined in any manner and in any number to produce thePS4 variant polypeptides disclosed herein.

Thus, we disclose PS4 variant polypeptides based on a wild type PS4parent sequence, preferably, a Pseudomonas saccharophilia non-maltogenicexoamylase having a sequence shown as SEQ ID NO: 1. Thus, the PS4variant polypeptide may comprise a sequence shown in SEQ ID NO: 1, butwith any one or more mutations at the positions set out above. The PS4variant polypeptides may also be based on a wild type Pseudomonasstutzeri non-maltogenic enzyme sequence shown as SEQ ID NO: 7 below butwith any one or more mutations at the positions set out above.

While PS4 variant polypeptides may be based on wild type PS4 parentsequences, in preferred embodiments PS4 variant polypeptides are basedon engineered (or mutated) versions of wild type PS4 parent sequences.Thus, such embodiments, the parent sequences comprise already mutatedPS4 sequences. Such sequences may be made de novo, or by mutating a basesequence, one or more times, as described in more detail below.

Positions 121, 161 and/or 223

The PS4 variant polypeptide may comprise a wild type sequence, or mutantsequence being a variant of a wild type sequence, such as an alreadymutated sequence, with substitutions at one more positions such as 121,161 and 223.

Thus, in general the subject mutation or mutations at the relevantposition or positions may advantageously be combined with a singleadditional mutation at one, two or all of positions 121, 161 or 223.

The position 121 substitution, where present, is preferably selectedfrom the group consisting of: 121F, 121Y, 121W, 121H, 121A, 121M, 121G,121S, 121T, 121D, 121E, 121L, 121K and 121V. Preferably, the position121 substitution is 121F, 121Y or 121W. The position 161 substitution,where present, is preferably 161A, more preferably S161A. Where position161 is mutated, a further mutation at position 160 may also be present,preferably 160D, more preferably E160D.

The position 223 substitution, where present, is preferably selectedfrom the group consisting of: 223K, 223E, 223V, 223R, 223A, 223P and223D. More preferably, the 223 substitution is 223E or 223K. Inparticularly preferred embodiments, the further substitution orsubstitutions are selected from the group consisting of: G121F, G121Y,G121W, 161A, 223E and 223K.

In a particularly preferred embodiment, the PS4 variant polypeptidecomprises at least the three following substitutions 121F/Y/W, 161A,223E/K. Other mutations may be included, as set out below.

Positions 146, 157, 158, 198, 229, 303, 306, 309, 316 and 353

The PS4 variant polypeptide may comprise a wild type sequence, or mutantsequence being a variant of a wild type sequence, such as an alreadymutated sequence—including a PS4 variant polypeptide having a mutationat one or more of positions 121, 161 and 223—with substitutions at onemore positions such as positions 146, 157, 158, 198, 229, 303, 306, 309,316 and 353.

Positions 26, 70, 145, 188, 272 and 339

The PS4 variant polypeptide may comprise a wild type sequence, or mutantsequence being a variant of a wild type sequence, such as an alreadymutated sequence—including a PS4 variant polypeptide having a mutationat one or more of positions 121, 161, 223, 146, 157, 158, 198, 229, 303,306, 309, 316 and 353.—with substitutions at one more positions such as26, 70, 145, 188, 272 and 339.

Thus, in general the subject mutation or mutations at the relevantposition or positions may advantageously be combined with a singleadditional mutation at one, two or all of positions 26, 70, 145, 188272and 339.

The PS4 variant polypeptide may comprise a substitution at position 26,preferably 26E or 26E. More preferably, the position 26 substitutioncomprises N26E or N26D. Most preferably, the position 26 substitutioncomprises N26E.

The PS4 variant polypeptide may comprise a substitution at position 70,preferably 70D. More preferably, the position 26 substitution comprisesG70D.

The PS4 variant polypeptide may comprise a substitution at position 145,preferably 145D. More preferably, the position 145 substitutioncomprises N145D.

The PS4 variant polypeptide may comprise a substitution at position 188,preferably 188S, 188T or 188H. More preferably, the position 188substitution comprises G188S, G188T or G188H. Most preferably, theposition 188 substitution comprises G188S or G188H.

The PS4 variant polypeptide may comprise a substitution at position 272,preferably 272Q. More preferably, the position 272 substitutioncomprises H272Q.

The PS4 variant polypeptide may comprise a substitution at positionW339A, preferably 339A or 339E. More preferably, the position 339substitution comprises W339A or W339E

PS4 Variant Polypeptides Based on Variant Sequences

Combinations with Positions 33, 34, 121, 134, 141, 157, 178, 179, 223,307 and/or 334

In some embodiments, the parent sequences comprise mutations at one ormore of, preferably all, of positions 33, 34, 121, 134, 141, 157, 178,179, 223, 307 and 334 (and accordingly the PS4 variant polypeptides willalso contain the relevant corresponding mutations).

In such embodiments, the mutations are preferably one or more of;preferably all of: N33Y, D34N, G121D, G134R, A141P, I157L, L178F, A179T,G223A, H307L and S334P.

In such embodiments, the PS4 variant polypeptide may conveniently bederivable from a Pseudomonas saccharophila non-maltogenic enzymesequence comprising a sequence PSac-D34 (SEQ ID NO: 2).

Combinations with Positions 33, 34, 121, 134, 141, 157, 178, 179, 223,307 and 334, 121, 161 and/or 223

PS4 variant polypeptides may further comprise mutations at any ofpositions 33, 34, 121, 134, 141, 157, 178, 179, 223, 307 and 334 incombination with mutations at any of positions 121, 161 and/or 223.Thus, we disclose a PS4 variant polypeptide comprising any combinationof: (a) any one or more of mutations at residues 33, 34, 121, 134, 141,157, 178, 179, 223, 307 and 334, preferably N33Y, D34N, G121D, G134R,A141P, I157L, L178F, A179T, G223A, H307L and S334P; (b) any one or moreof mutations at positions 121, 161 or 223, preferably 121F, 121Y, 121W,161A, 223E or 223K, more preferably 121F, 161A or 223E, and (c) any oneor more of mutations at positions 146, 157, 158, 198, 229, 303, 306,309, 316 or 353, preferably 146G, 146M, 157M, 158T, 158A, 158S, 198W,198F, 229P, 303E, 303D, 306T, 306G, 309P, 316S, 316P, 316K, 316Q or353T, more preferably 146G, 157M, 158T, 198W, 229P, 303E, 303D, 306T,306G, 309P, 316S, 316P or 353T.

In some preferred embodiments, we provide a PS4 variant polypeptidecomprising all of the following mutations set out in (a) above incombination with one or more of mutations set out in (c) above. In otherpreferred embodiments, we provide a PS4 variant polypeptide comprisingall of the following mutations set out in (a) above in combination withall of the mutations set out in (b) above, in combination with any oneor more of the mutations set out in (c) above.

PS4 Variant Polypeptides Based on pMD96

In embodiments where mutations in all of (a) and (b) are included, thePS4 variant polypeptide may conveniently be derivable from a Pseudomonassaccharophila non-maltogenic enzyme sequence comprising a sequence pMD96(SEQ ID NO: 14).

Thus, we specifically provide PS4 variant polypeptides set out inExamples 12 to 20, having sequences SEQ ID NO: 15 to 28. As shown in theExamples, each of these polypeptides has one or more improved propertiescompared to its parent.

Other Combinations

Furthermore, parent sequences comprising mutations at other positions,for example, any one or more of 134, 141, 157, 223, 307 and 334 may alsobe used. Optionally, these may include mutations at one or both ofpositions 33 and 34.

Thus, the parent sequence may comprise one or more mutations atpositions selected from the group consisting of: 134, 141, 157, 223,307, 334 and optionally 33 and 34, (and accordingly of course the PS4variant polypeptides will also contain the relevant correspondingmutations).

In some embodiments, the parent polypeptides comprise substitutionsarginine at position 134, proline at position 141 and proline atposition 334, e.g., G134R, A141P and S334P.

In further embodiments, the parent polypeptide further comprises amutation at position 121. The parent polypeptide may further comprise amutation at position 178. It may further comprise a mutation at position179. It may yet further comprise a mutation at position 87. Therespective particularly preferred substitutions are preferably 121D,more preferably G121D, preferably 178F, more preferably L178F,preferably 179T, more preferably A179T and preferably 87S, morepreferably G87S.

The residues at these positions may be substituted by a number ofresidues, for example I157V or 1157N or G223L or G223I or G223S or G223Tor H307I or H307V or D34G or D34A or D34S or D34T or A179V. However, theparent polypeptides preferably comprise the substitutions I157L, G223A,H307L, L178F and A179T (optionally N33Y, D34N).

In a highly preferred embodiment, the PS4 variant polypeptides comprisea substitution at any one or more of positions 146, 157, 158, 198, 229,303, 306, 309, 316 and 353, preferably 146G, 157M, 158T, 198W, 229P,303E, 303D, 306T, 306G, 309P, 316S, 316P or 353T, as well as one or moreof the following substitutions: G134R, A141P, I157L, G223A, H307L,S334P, N33Y and D34N, together with one or both of L178F and A179T.

PS4 Variant Polypeptides Based on PSac-D20

A PS4 variant may be based on a Pseudomonas saccharophila non-maltogenicparent enzyme sequence PSac-D20 (SEQ ID NO: 3).

In such an embodiment, the PS4 variant polypeptides comprise asubstitution any one or more of positions 146, 157, 158, 198, 229, 303,306, 309, 316 and 353, preferably 146G, 157M, 158T, 198W, 229P, 303E,303D, 306T, 306G, 309P, 316S, 316P or 353T, as well as one or more ofthe following substitutions: G134R, A141P, I157L, G223A, H307L, S334P,N33Y, D34N and G121D, together with one or both of L178F and A179T.

PS4 Variant Polypeptides Based on PSac-D14

A PS4 variant may be based on a Pseudomonas saccharophila non-maltogenicparent enzyme sequence PSac-D14 (SEQ ID NO: 4).

In such an embodiment, therefore the PS4 variant polypeptides comprise asubstitution at any one or more of positions 146, 157, 158, 198, 229,303, 306, 309, 316 and 353, preferably 146G, 157M, 158T, 198W, 229P,303E, 303D, 306T, 306G, 309P, 316S, 316P or 353T, as well as one or moreof the following substitutions: G134R, A141P, I157L, G223A, H307L,S334P, N33Y, D34N, G121D and G87S, together with one or both of L178Fand A179T.

PS4 Variant Polypeptides Based on pMD55

A PS4 variant may be based on a Pseudomonas saccharophila non-maltogenicparent enzyme sequence pMD55 (SEQ ID NO: 13).

In such an embodiment, the PS4 variant polypeptides comprise asubstitution any one or more of positions 146, 157, 158, 198, 229, 303,306, 309, 316 and 353, preferably 146G, 157M, 158T, 198W, 229P, 303E,303D, 306T, 306G, 309P, 316S, 316P or 353T, as well as one or more ofthe following substitutions: G134R, A141P, I157L, G223A, H307L, S334P,N33Y, D34N, G121F and G87S, together with one or both of L178F andA179T. The PS4 variant polypeptide may be derivable from a parentpolypeptide having such substitutions.

PS4 Variant Polypeptides Based on Pseudomonas stutzeri ParentPolypeptides

In some embodiments, the PS4 variants are derived from a Pseudomonasstutzeri non-maltogenic enzyme sequence, preferably shown as SEQ ID NO:7 below.

Accordingly, the PS4 variant polypeptide may be derivable from asequence PStu-D34 (SEQ ID NO: 8). We further disclose PS4 variantpolypeptides based on Pseudomonas stutzeri non-maltogenic enzymesequence and including 0121 and/or G87 substitutions. These may comprisethe following substitutions: G134R, A141P, I157L, G223A, H307L, S334P,N33Y, D34N and G121D, together with one or both of L178F and A179T, aswell as PS4 variant polypeptides comprising the following substitutions:G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, G121D and G87S,together with one or both of L178F and A179T.

Therefore, a PS4 variant polypeptide may be derived from a Pseudomonasstutzeri non-maltogenic enzyme parent sequence, which parent sequencemay have a sequence PStu-D20 (SEQ ID NO: 9), comprising G121D, or asequence PStu-D14 (SEQ ID NO: 10), further comprising G87S.

PS4 Variant Nucleic Acids

We also describe PS4 nucleic acids having sequences which correspond toor encode the alterations in the PS4 variant polypeptide sequences, foruse in producing such polypeptides for the purposes described here.Thus, we provide nucleic acids capable of encoding any polypeptidesequence set out in this document.

The skilled person will be aware of the relationship between nucleicacid sequence and polypeptide sequence, in particular, the genetic codeand the degeneracy of this code, and will be able to construct such PS4nucleic acids without difficulty. For example, he will be aware that foreach amino acid substitution in the PS4 variant polypeptide sequence,there may be one or more codons which encode the substitute amino acid.Accordingly, it will be evident that, depending on the degeneracy of thegenetic code with respect to that particular amino acid residue, one ormore PS4 nucleic acid sequences may be generated corresponding to thatPS4 variant polypeptide sequence. Furthermore, where the PS4 variantpolypeptide comprises more than one substitution, for exampleA141P/G223A, the corresponding PS4 nucleic acids may comprise pairwisecombinations of the codons which encode respectively the two amino acidchanges.

The PS4 variant nucleic acid sequences may be derivable from parentnucleic acids which encode any of the parent polypeptides describedabove. In particular, parent nucleic acids may comprise wild typesequences, e.g., SEQ ID NO: 6 or SEQ ID NO: 12. The PS4 variant nucleicacids may therefore comprise nucleic acids encoding wild typenon-maltogenic exoamylases, but which encode another amino acid at therelevant position instead of the wild type amino acid residue. The PS4variant nucleic acid sequences may also comprise wild type sequenceswith one or more mutations, e.g., which encode parent polypeptidesdescribed above under “Combinations”.

It will be understood that nucleic acid sequences which are notidentical to the particular PS4 variant nucleic acid sequences, but arerelated to these, will also be useful for the methods and compositionsdescribed here, such as a variant, homologue, derivative or fragment ofa PS4 variant nucleic acid sequence, or a complement or a sequencecapable of hybridising thereof. Unless the context dictates otherwise,the term “PS4 variant nucleic acid” should be taken to include each ofthese entities listed above.

Mutations in amino acid sequence and nucleic acid sequence may be madeby any of a number of techniques, as known in the art. Variant sequencesmay easily be made using any of the known mutagenesis techniques, forexample, site directed mutagenesis using PCR with appropriateoligonucleotide primers, 5′ add-on mutagenesis, mismatched primermutagenesis, etc. Alternatively, or in addition, the PS4 variant nucleicacid sequences may be made de novo.

In particularly preferred embodiments, the mutations are introduced intoparent sequences by means of PCR (polymerase chain reaction) usingappropriate primers, as illustrated in the Examples. It is thereforepossible to alter the sequence of a polypeptide by introducing anydesired amino acid substitutions into a parent polypeptide, preferablyhaving non-maltogenic exoamylase activity, such as into a Pseudomonassaccharophilia or a Pseudomonas stutzeri exoamylase sequence at aminoacid or nucleic acid level, as described. We describe a method in whichthe sequence of a non-maltogenic exoamylase is altered by altering thesequence of a nucleic acid which encodes the non-maltogenic exoamylase.

However, it will of course be appreciated that the PS4 variantpolypeptide does not need in fact to be actually derived from a wildtype polypeptide or nucleic acid sequence by, for example, step by stepmutation. Rather, once the sequence of the PS4 variant polypeptide isestablished, the skilled person can easily make that sequence from thewild type with all the mutations, via means known in the art forexample, using appropriate oligonucleotide primers and PCR. In fact, thePS4 variant polypeptide can be made de novo with all its mutations,through, for example, peptide synthesis methodology.

In general, however, the PS4 variant polypeptides and/or nucleic acidsare derived or derivable from a “precursor” sequence. The term“precursor” as used herein means an enzyme that precedes the enzymewhich is modified according to the methods and compositions describedhere. A precursor therefore includes an enzyme used to produce amodified enzyme. Thus, the precursor may be an enzyme that is modifiedby mutagenesis as described elsewhere in this document. Likewise, theprecursor may be a wild type enzyme, a variant wild type enzyme or analready mutated enzyme.

The PS4 variant polypeptides and nucleic acids may be produced by anymeans known in the art. Specifically, they may be expressed fromexpression systems, which may be in vitro or in vivo in nature.Specifically, we describe plasmids and expression vectors comprising PS4nucleic acid sequences, preferably capable of expressing PS4 variantpolypeptides. Cells and host cells which comprise and are preferablytransformed with such PS4 nucleic acids, plasmids and vectors are alsodisclosed, and it should be made clear that these are also encompassedin this document.

In preferred embodiments, the PS4 variant polypeptide sequence is usedas a food additive in an isolated form. The term “isolated” means thatthe sequence is at least substantially free from at least one othercomponent with which the sequence is naturally associated in nature andas found in nature. In one aspect, preferably the sequence is in apurified form. The term “purified” means that the sequence is in arelatively pure state—e.g. at least about 90% pure, or at least about95% pure or at least about 98% pure.

Position Numbering

All positions referred to in the present document by numbering refer tothe numbering of a Pseudomonas saccharophilia exoamylase referencesequence shown below (SEQ ID NO: 1):

1 DQAGKSPAGV RYHGGDEIIL QGFHWNVVRE APNDWYNILR QQASTIAADG FSAIWMPVPW 61RDFSSWTD

G KSGGGEGYFW HDFNKNGRYG SDAQLRQAAG ALGGAGVKVL YDVVPNHMNR 121GYPDKEINLP AGQGFWRNDC 

DPGNYPNDC DDGDRFIGGE SDLNTGHPQI YGMFRDELAN 181LRSGYGAGGF RFDFVRGYAP ERVDSWMSDS ADSSFCVGEL WK

PSEYPSW DWRNTASWQQ 241 IIKDWSDRAK CPVFDFALKE RMQNGSV

DW KHGLNGNPDP RWREVAVTFV DNHDTGYSPG 301 QNGGQHHWAL QD

LIRQAYA YILTSPGTPV VYWSHMYDWG YGDFIRQLIQ VRRTAGVRAD 361SAISFHSGYS GLVATVSGSQ QTLVVALNSD LANPGQVA

 SFSEAVNASN GQVRVWRSGS 421GDGGGNDGGE GGLVNVNFRC DNGVTQMGDS VYAVGNVSQL GNWSPASAVR LTDTSSYPTW 481KGSIALPDGQ NVEWKCLIRN EADATLVRQW QSGGNNQVQA AAGASTSGSF

The reference sequence is derived from the Pseudomonas saccharophiliasequence having SWISS-PROT accession number P22963, but without thesignal sequence MSHILRAAVLAAVLLPFPALA.

The C-terminal starch binding domain EGGLVNVNFR CDNGVTQMGD SVYAVGNVSQLGNWSPASAV RLTDTSSYPT WKGSIALPDG QNVEWKCLIR NEADATLVRQ WQSGGNNQVQAAAGASTSGS F may optionally be disregarded. Alternatively, it may beincluded.

In the context of the present description a specific numbering of aminoacid residue positions in PS4 exoamylase enzymes is employed. In thisrespect, by alignment of the amino acid sequences of various knownexoamylases it is possible to unambiguously allot a exoamylase aminoacid position number to any amino acid residue position in anyexoamylase enzyme, the amino acid sequence of which is known. Using thisnumbering system originating from for example the amino acid sequence ofthe exoamylase obtained from Pseudomonas saccharophilia, aligned withamino acid sequences of a number of other known exoamylase, it ispossible to indicate the position of an amino acid residue in aexoamylase unambiguously.

Therefore, the numbering system, even though it may use a specificsequence as a base reference point, is also applicable to all relevanthomologous sequences. For example, the position numbering may be appliedto homologous sequences from other Pseudomonas species, or homologoussequences from other bacteria. Preferably, such homologous have 60% orgreater homology, for example 70% or more, 80% or more, 90% or more or95% or more homology, with the reference sequence SEQ ID NO: 1 above, orthe sequences having SWISS-PROT accession numbers P22963 or P13507,preferably with all these sequences. Sequence homology between proteinsmay be ascertained using well known alignment programs and hybridisationtechniques described herein. Such homologous sequences, as well as thefunctional equivalents described below, will be referred to in thisdocument as the “PS4 Family”.

Furthermore, and as noted above, the numbering system used in thisdocument makes reference to a reference sequence SEQ ID NO: 1, which isderived from the Pseudomonas saccharophilia sequence having SWISS-PROTaccession number P22963, but without the signal sequenceMSHILIRAAVLAAVLLPFPALA. This signal sequence is located N terminal ofthe reference sequence and consists of 21 amino acid residues.Accordingly, it will be trivial to identify the particular residues tobe mutated or substituted in corresponding sequences comprising thesignal sequence, or indeed, corresponding sequences comprising any otherN- or C-terminal extensions or deletions. In relation to N-terminaladditions or deletions, all that is required is to offset the positionnumbering by the number of residues inserted or deleted. For example,position 1 in SEQ ID NO: 1 corresponds to position 22 in a sequence withthe signal sequence.

Parent Enzyme/Polypeptide

The PS4 variant polypeptides are derived from, or are variants of,another sequence, known as a “parent enzyme”, a “parent polypeptide” ora “parent sequence”.

The term “parent enzyme” as used in this document means the enzyme thathas a close, preferably the closest, chemical structure to the resultantvariant, i.e., the PS4 variant polypeptide or nucleic acid. The parentenzyme may be a precursor enzyme (i.e. the enzyme that is actuallymutated) or it may be prepared de novo. The parent enzyme may be a wildtype enzyme, or it may be a wild type enzyme comprising one or moremutations.

The term “precursor” as used herein means an enzyme that precedes theenzyme which is modified to produce the enzyme. Thus, the precursor maybe an enzyme that is modified by mutagenesis. Likewise, the precursormay be a wild type enzyme, a variant wild type enzyme or an alreadymutated enzyme.

The term “wild type” is a term of the art understood by skilled personsand means a phenotype that is characteristic of most of the members of aspecies occurring naturally and contrasting with the phenotype of amutant. Thus, in the present context, the wild type enzyme is a form ofthe enzyme naturally found in most members of the relevant species.Generally, the relevant wild type enzyme in relation to the variantpolypeptides described here is the most closely related correspondingwild type enzyme in terms of sequence homology. However, where aparticular wild type sequence has been used as the basis for producing avariant PS4 polypeptide as described here, this will be thecorresponding wild type sequence regardless of the existence of anotherwild type sequence that is more closely related in terms of amino acidsequence homology.

The parent enzyme is preferably a polypeptide which preferably exhibitsnon-maltogenic exoamylase activity. Preferably, the parent enzyme is anon-maltogenic exoamylase itself. For example, the parent enzyme may bea Pseudomonas saccharophila non-maltogenic exoamylase, such as apolypeptide having SWISS-PROT accession number P22963, or a Pseudomonasstutzeri non-maltogenic exoamylase, such as a polypeptide havingSWISS-PROT accession number P13507.

Other members of the PS4 family may be used as parent enzymes; such “PS4family members” will generally be similar to, homologous to, orfunctionally equivalent to either of these two enzymes, and may beidentified by standard methods, such as hybridisation screening of asuitable library using probes, or by genome sequence analysis.

In particular, functional equivalents of either of these two enzymes, aswell as other members of the “PTS4 family” may also be used as startingpoints or parent polypeptides for the generation of PS4 variantpolypeptides as described here.

A “functional equivalent” of a protein means something that shares oneor more, preferably substantially all, of the functions of that protein.Preferably, such functions are biological functions, preferablyenzymatic functions, such as amylase activity, preferably non-maltogenicexoamylase activity. In relation to a parent enzyme, the term“functional equivalent” preferably means a molecule having similar oridentical function to a parent molecule. The parent molecule may be aPseudomonas saccharophila non-maltogenic exoamylase or a Pseudomonasstutzeri non-maltogenic exoamylase or a polypeptide obtained from othersources.

The term “functional equivalent” in relation to a parent enzyme being aPseudomonas saccharophila non-maltogenic exoamylase, such as apolypeptide having SWISS-PROT accession number P22963, or a Pseudomonasstutzeri non-maltogenic exoamylase, such as a polypeptide havingSWISS-PROT accession number P13507 means that the functional equivalentcould be obtained from other sources. The functionally equivalent enzymemay have a different amino acid sequence but will have non-maltogenicexoamylase activity. Examples of assays to determine functionality aredescribed herein and are known to one skilled in the art.

In highly preferred embodiments, the functional equivalent will havesequence homology to either of the Pseudomonas saccharophila andPseudomonas stutzeri non-maltogenic exoamylases mentioned above,preferably both. The functional equivalent may also have sequencehomology with any of the sequences set out as SEQ ID NOs: 1 to 14,preferably SEQ ID NO: 1 or SEQ ID NO: 7 or both. Sequence homologybetween such sequences is preferably at least 60%, preferably 65% ormore, preferably 75% or more, preferably 80% or more, preferably 85% ormore, preferably 90% or more, preferably 95% or more. Such sequencehomologies may be generated by any of a number of computer programsknown in the art, for example BLAST or FASTA, etc. A suitable computerprogram for carrying out such an alignment is the GCG Wisconsin Bestfitpackage (University of Wisconsin, U.S.A; Devereux et al., 1984, NucleicAcids Research 12:387). Examples of other software than can performsequence comparisons include, but are not limited to, the BLAST package(see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul et al., 1990,J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools.Both BLAST and FASTA are available for offline and online searching (seeAusubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferredto use the GCG Bestfit program.

In other embodiments, the functional equivalents will be capable ofspecifically hybridising to any of the sequences set out above. Methodsof determining whether one sequence is capable of hybridising to anotherare known in the art and are for example described in Sambrook, et al(supra) and Ausubel, F. M. et al. (supra). In highly preferredembodiments, the functional equivalents will be capable of hybridisingunder stringent conditions, e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl,0.015 M Na₃ Citrate pH 7.0}.

For example, functional equivalents which have sequence homology toPseudomonas saccharophila and Pseudomonas stutzeri non-maltogenicexoamylases are suitable for use as parent enzymes. Such sequences maydiffer from the Pseudomonas saccharophila sequence at any one or morepositions. Furthermore, non-maltogenic exoamylases from other strains ofPseudomonas spp, such as ATCC17686, may also be used as a parentpolypeptide. The PS4 variant polypeptide residues may be inserted intoany of these parent sequences to generate the variant PS4 polypeptidesequences.

It will be understood that where it is desired for PS4 variantpolypeptides to additionally comprise one or more mutations, as set outabove, corresponding mutations may be made in the nucleic acid sequencesof the functional equivalents of Pseudomonas spp non-maltogenicexoamylase, as well as other members of the “PS4 family”, in order thatthey may be used as starting points or parent polypeptides for thegeneration of PS4 variant polypeptides as described here.

Specifically included within the term “PS4 variant polypeptides” are thepolypeptides disclosed in:

U.S. 60/485,413, 60/485,539 and 60/485,616; PCT/US2004/021723 andPCT/US2004/021739; U.S. Ser. No. 10/886,905 and 10/866,903; U.S.60/608,919; U.S. 60/612,407; U.S. 60/485,539; PCT/IB2004/002487; U.S.Ser. No. 10/886,023; U.S. Ser. No. 10/886,505, U.S. Ser. No. 10/886,527and U.S. Ser. No. 10/886,504; U.S. Ser. No. 10/947,612.

Such polypeptides are suitable for use in the applications describedherein, in particular, as food additives, to treat starch as described,to prepare a food product, to make a bakery product, for the formulationof improver compositions, for the formulation of combinations, etc.

Modification of Parent Sequences

The parent enzymes may be modified at the amino acid level or thenucleic acid level to generate the PS4 variant sequences described here.Therefore, we provide for the generation of PS4 variant polypeptides byintroducing one or more corresponding codon changes in the nucleotidesequence encoding a non-maltogenic exoamylase polypeptide.

The nucleic acid numbering should preferably be with reference to theposition numbering of a Pseudomonas saccharophilia exoamylase nucleotidesequence shown as SEQ ID NO: 6. Alternatively, or in addition, referencemay be made to the sequence with GenBank accession number X16732. Inpreferred embodiments, the nucleic acid numbering should be withreference to the nucleotide sequence shown as SEQ ID NO: 6. However, aswith amino acid residue numbering, the residue numbering of thissequence is to be used only for reference purposes only. In particular,it will be appreciated that the above codon changes can be made in anyPS4 family nucleic acid sequence. For example, sequence changes can bemade to a Pseudomonas saccharophila or a Pseudomonas stutzerinon-maltogenic exoamylase nucleic acid sequence (e.g., X16732, SEQ IDNO: 6 or M24516, SEQ ID NO: 12).

The parent enzyme may comprise the “complete” enzyme, i.e., in itsentire length as it occurs in nature (or as mutated), or it may comprisea truncated form thereof. The PS4 variant derived from such mayaccordingly be so truncated, or be “full-length”. The truncation may beat the N-terminal end, or the C-terminal end, preferably the C-terminalend. The parent enzyme or PS4 variant may lack one or more portions,such as sub-sequences, signal sequences, domains or moieties, whetheractive or not etc. For example, the parent enzyme or the PS4 variantpolypeptide may lack a signal sequence, as described above.Alternatively, or in addition, the parent enzyme or the PS4 variant maylack one or more catalytic or binding domains.

In highly preferred embodiments, the parent enzyme or PS4 variant maylack one or more of the domains present in non-maltogenic exoamylases,such as the starch binding domain. For example, the PS4 polypeptides mayhave only sequence up to position 429, relative to the numbering of aPseudomonas saccharophilia non-maltogenic exoamylase shown as SEQ IDNO: 1. It is to be noted that this is the case for the PS4 variantspSac-d34, pSac-D20 and pSac-D14.

In other embodiments, the parent enzyme or PS4 variant may comprise a e“complete” enzyme, i.e., in its entire length as it occurs in nature (oras mutated), together with one or more additional amino acid sequencesat the N terminus or C terminus. For example, the parent enzyme or PS4variant polypeptide may comprise a single extra amino acid residue atthe C terminus or N terminus, e.g., M, A, G, etc. Preferably, theadditional amino acid residue is present at the N terminus. Where one ormore additional residues is included, the position numbering will beoffset by the length of the addition.

Amylase

The PS4 variant polypeptides generally comprise amylase activity.

The term “amylase” is used in its normal sense—e.g. an enzyme that isinter alia capable of catalysing the degradation of starch. Inparticular they are hydrolases which are capable of cleaving α-D-(1→4)O-glycosidic linkages in starch.

Amylases are starch-degrading enzymes, classified as hydrolases, whichcleave α-D-(1→4) β-glycosidic linkages in starch. Generally, β-amylases(E.C. 3.2.1.1, α-D-(1→4)-glucan glucanohydrolase) are defined asendo-acting enzymes cleaving α-D-(1→4) O-glycosidic linkages within thestarch molecule in a random fashion. In contrast, the exo-actingamylolytic enzymes, such as β-amylases (E.C. 3.2.1.2, α-D-(1→4)-glucanmaltohydrolase), and some product-specific amylases like maltogenicalpha-amylase (E.C. 3.2.1.133) cleave the starch molecule from thenon-reducing end of the substrate. β-Amylases, α-glucosidases (E.C.3.2.1.20, α-D-glucoside glucohydrolase), glucoamylase (E.C. 3.2.1.3,α-D-(1→4)-glucan glucohydrolase), and product-specific amylases canproduce malto-oligosaccharides of a specific length from starch.

Non-Maltogenic Exoamylase

The PS4 variant polypeptides described in this document are derived from(or variants of) polypeptides which preferably exhibit non-maltogenicexoamylase activity. Preferably, these parent enzymes are non-maltogenicexoamylases themselves. The PS4 variant polypeptides themselves inhighly preferred embodiments also exhibit non-maltogenic exoamylaseactivity.

In highly preferred embodiments, the term “non-maltogenic exoamylaseenzyme” as used in this document should be taken to mean that the enzymedoes not initially degrade starch to substantial amounts of maltose asanalysed in accordance with the product determination procedure asdescribed in this document.

In highly preferred embodiments, the non-maltogenic exoamylase comprisesan exo-maltotetraohydrolase. Exo-maltotetraohydrolase (E.C.3.2.1.60) ismore formally known as glucan 1,4-alpha-maltotetrahydrolase. This enzymehydrolyses 1,4-alpha-D-glucosidic linkages in amylaceous polysaccharidesso as to remove successive maltotetraose residues from the non-reducingchain ends.

Non-maltogenic exoamylases are described in detail in U.S. Pat. No.6,667,065, hereby incorporated by reference.

Assays for Non-Maltogenic Exoamylase Activity

The following system is used to characterize polypeptides havingnon-maltogenic exoamylase activity which are suitable for use accordingto the methods and compositions described here. This system may forexample be used to characterise the PS4 parent or variant polypeptidesdescribed here.

By way of initial background information, waxy maize amylopectin(obtainable as WAXILYS 200 from Roquette, France) is a starch with avery high amylopectin content (above 90%). 20 mg/ml of waxy maize starchis boiled for 3 min. in a buffer of 50 mM MES(2-(N-morpholino)ethanesulfonic acid), 2 mM calcium chloride, pH 6.0 andsubsequently incubated at 50° C. and used within half an hour.

One unit of the non-maltogenic exoamylase is defined as the amount ofenzyme which releases hydrolysis products equivalent to 1 μmol ofreducing sugar per min. when incubated at 50 degrees C. in a test tubewith 4 ml of 10 mg/ml waxy maize starch in 50 mM MES, 2 mM calciumchloride, pH 6.0 prepared as described above. Reducing sugars aremeasured using maltose as standard and using the dinitrosalicylic acidmethod of Bernfeld, Methods Enzymol., (1954), 1, 149-158 or anothermethod known in the art for quantifying reducing sugars.

The hydrolysis product pattern of the non-maltogenic exoamylase isdetermined by incubating 0.7 units of non-maltogenic exoamylase for 15or 300 min. at 50° C. in a test tube with 4 ml of 10 mg/ml waxy maizestarch in the buffer prepared as described above. The reaction isstopped by immersing the test tube for 3 min. in a boiling water bath.

The hydrolysis products are analyzed and quantified by anion exchangeHPLC using a Dionex PA 100 column with sodium acetate, sodium hydroxideand water as eluents, with pulsed amperometric detection and with knownlinear maltooligosaccharides of from glucose to maltoheptaose asstandards. The response factor used for maltooctaose to maltodecaose isthe response factor found for maltoheptaose.

Preferably, the PS4 variant polypeptides have non-maltogenic exoamylaseactivity such that if an amount of 0.7 units of said non-maltogenicexoamylase were to incubated for 15 minutes at a temperature of 50° C.at pH 6.0 in 4 ml of an aqueous solution of 10 mg preboiled waxy maizestarch per ml buffered solution containing 50 mM 2-(N-morpholino)ethanesulfonic acid and 2 mM calcium chloride then the enzyme would yieldhydrolysis product(s) that would consist of one or more linearmalto-oligosaccharides of from two to ten D-glucopyranosyl units andoptionally glucose; such that at least 60%, preferably at least 70%,more preferably at least 80% and most preferably at least 85% by weightof the said hydrolysis products would consist of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units,preferably of linear maltooligosaccharides consisting of from four toeight D-glucopyranosyl units.

For ease of reference, and for the present purposes, the feature ofincubating an amount of 0.7 units of the non-maltogenic exoamylase for15 minutes at a temperature of 50° C. at pH 6.0 in 4 ml of an aqueoussolution of 10 mg preboiled waxy maize starch per ml buffered solutioncontaining 50 mM 2-(N-morpholino)ethane sulfonic acid and 2 mM calciumchloride, may be referred to as the “Waxy Maize Starch Incubation Test”.

Thus, alternatively expressed, preferred PS4 variant polypeptides whichare non-maltogenic exoamylases are characterised as having the abilityin the waxy maize starch incubation test to yield hydrolysis product(s)that would consist of one or more linear malto-oligosaccharides of fromtwo to ten D-glucopyranosyl units and optionally glucose; such that atleast 60%, preferably at least 70%, more preferably at least 80% andmost preferably at least 85% by weight of the said hydrolysis product(s)would consist of linear maltooligosaccharides of from three to tenD-glucopyranosyl units, preferably of linear maltooligosaccharidesconsisting of from four to eight D-glucopyranosyl units.

The hydrolysis products in the waxy maize starch incubation test mayinclude one or more linear malto-oligosaccharides of from two to tenD-glucopyranosyl units and optionally glucose. The hydrolysis productsin the waxy maize starch incubation test may also include otherhydrolytic products. Nevertheless, the % weight amounts of linearmaltooligosaccharides of from three to ten D-glucopyranosyl units arebased on the amount of the hydrolysis product that consists of one ormore linear malto-oligosaccharides of from two to ten D-glucopyranosylunits and optionally glucose. In other words, the % weight amounts oflinear maltooligosaccharides of from three to ten D-glucopyranosyl unitsare not based on the amount of hydrolysis products other than one ormore linear malto-oligosaccharides of from two to ten D-glucopyranosylunits and glucose.

The hydrolysis products can be analysed by any suitable means. Forexample, the hydrolysis products may be analysed by anion exchange HPLCusing a Dionex PA 100 column with pulsed amperometric detection andwith, for example, known linear maltooligosaccharides of from glucose tomaltoheptaose as standards.

For ease of reference, and for the present purposes, the feature ofanalysing the hydrolysis product(s) using anion exchange HPLC using aDionex PA 100 column with pulsed amperometric detection and with knownlinear maltooligosaccharides of from glucose to maltoheptaose used asstandards, can be referred to as “analysing by anion exchange”. Ofcourse, and as just indicated, other analytical techniques wouldsuffice, as well as other specific anion exchange techniques.

Thus, alternatively expressed, a preferred PS4 variant polypeptide isone which has non-maltogenic exoamylase such that it has the ability ina waxy maize starch incubation test to yield hydrolysis product(s) thatwould consist of one or more linear malto-oligosaccharides of from twoto ten D-glucopyranosyl units and optionally glucose, said hydrolysisproducts being capable of being analysed by anion exchange; such that atleast 60%, preferably at least 70%, more preferably at least 80% andmost preferably at least 85% by weight of the said hydrolysis product(s)would consist of linear maltooligosaccharides of from three to tenD-glucopyranosyl units, preferably of linear maltooligosaccharidesconsisting of from four to eight D-glucopyranosyl units.

As used herein, the term “linear malto-oligosaccharide” is used in thenormal sense as meaning 2-10 units of α-D-glucopyranose linked by anα-(1→4) bond.

In highly preferred embodiments, the PS4 polypeptides described herehave improved exoamylase activity, preferably non-maltogenic exoamylaseactivity, when compared to the parent polypeptide, preferably whentested under the same conditions. In particular, in highly preferredembodiments, the PS4 variant polypeptides have 10% or more, preferably20% or more, preferably 50% or more, exoamylase activity compared totheir parents, preferably when measured in a waxy maize starch test.

The hydrolysis products can be analysed by any suitable means. Forexample, the hydrolysis products may be analysed by anion exchange HPLCusing a Dionex PA 100 column with pulsed amperometric detection andwith, for example, known linear maltooligosaccharides of from glucose tomaltoheptaose as standards.

As used herein, the term “linear malto-oligosaccharide” is used in thenormal sense as meaning 2-20 units of α-D-glucopyranose linked by anα-(1→4) bond.

Improved Properties

The PS4 variants described here preferably have improved properties whencompared to their parent enzymes, such as any one or more of improvedthermostability, improved pH stability, or improved exo-specificity.

In particular, the PS4 variant polypeptides having mutations at position303, for example, 303E, 303D, 306T and 306G have increasedexo-specificity. Those having mutations at any of positions 146, 157,158, 198, 229, (preferably both 198 and 229), 309, 316, 316 and 353, forexample, 146G, 157M, 18T, 198W, 229P, (198W, 229P), 309P, 316S, 316P and353T display improved thermostability.

Without wishing to be bound by any particular theory, we believe thatthe mutations at the particular positions have individual and cumulativeeffects on the properties of a polypeptide comprising such mutations.

Thermostability and pH Stability

Preferably, the PS4 variant polypeptide is thermostable; preferably, ithas higher thermostability than its parent enzyme.

We therefore provide PS4 variant polypeptides which have a higherthermostability compared to the parent polypeptide or a wild typepolypeptide when tested under the same conditions. Specifically, weprovide for PS4 variant polypeptides comprising mutations at any one ormore of positions 121, 145, 146, 157, 158, 188, 198, 223, 229, 316, 353,more preferably any one or more of mutations 121A, 121D, 121F, 121H,121M, 121W, 121Y, 145D, 146G, 157M, 158T, 188H, 188S, 198W, 223A, 223E,223K, 223R, 223V, 229P, 316P, 316S, 353T.

In wheat and other cereals the external side chains in amylopectin arein the range of DP 12-19. Thus, enzymatic hydrolysis of the amylopectinside chains, for example, by PS4 variant polypeptides as describedhaving non-maltogenic exoamylase activity, can markedly reduce theircrystallisation tendencies.

Starch in wheat and other cereals used for baking purposes is present inthe form of starch granules which generally are resistant to enzymaticattack by amylases. Thus starch modification is mainly limited todamaged starch and is progressing very slowly during dough processingand initial baking until gelatinisation starts at about 60 C. As aconsequence hereof only amylases with a high degree of thermostabilityare able to modify starch efficiently during baking. And generally theefficiency of amylases is increased with increasing thermostability.That is because the more thermostable the enzyme is the longer time itcan be active during baking and thus the more antistaling effect it willprovide.

Accordingly, the use of PS4 variant polypeptides as described here whenadded to the starch at any stage of its processing into a food product,e.g., before during or after baking into bread can retard or impede orslow down the retrogradation. Such use is described in further detailbelow.

As used herein the term “thermostable” relates to the ability of theenzyme to retain activity after exposure to elevated temperatures.Preferably, the PS4 variant polypeptide is capable of degrading starchat temperatures of from about 55° C. to about 80° C. or more. Suitably,the enzyme retains its activity after exposure to temperatures of up toabout 95° C.

The thermostability of an enzyme such as a non-maltogenic exoamylase ismeasured by its half life. Thus, the PS4 variant polypeptides describedhere have half lives extended relative to the parent enzyme bypreferably 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% ormore, preferably at elevated temperatures of from 55° C. to about 95° C.or more, preferably at about 80° C.

As used here, the half life (t1/2) is the time (in minutes) during whichhalf the enzyme activity is inactivated under defined heat conditions.In preferred embodiments, the half life is assayed at 80 degrees C.Preferably, the sample is heated for 1-10 minutes at 80° C. or higher.The half life value is then calculated by measuring the residual amylaseactivity, by any of the methods described here. Preferably, a half lifeassay is conducted as described in more detail in the Examples.

Preferably, the PS4 variants described here are active during baking andhydrolyse starch during and after the gelatinization of the starchgranules which starts at temperatures of about 55° C. The morethermostable the non-maltogenic exoamylase is the longer time it can beactive and thus the more antistaling effect it will provide. However,during baking above temperatures of about 85° C., enzyme inactivationcan take place. If this happens, the non-maltogenic exoamylase may begradually inactivated so that there is substantially no activity afterthe baking process in the final bread. Therefore preferentially thenon-maltogenic exoamylases suitable for use as described have an optimumtemperature above 50° C. and below 98° C.

The thermostability of the PS4 variants described here can be improvedby using protein engineering to become more thermostable and thus bettersuited for the uses described here; we therefore encompass the use ofPS4 variants modified to become more thermostable by proteinengineering.

Preferably, the PS4 variant polypeptide is pH stable; more preferably,it has a higher pH stability than its cognate parent polypeptide. Asused herein the term “pH stable” relates to the ability of the enzyme toretain activity over a wide range of pHs. Preferably, the PS4 variantpolypeptide is capable of degrading starch at a pH of from about 5 toabout 10.5. In one embodiment, the degree of pH stability may be assayedby measuring the half life of the enzyme in specific pH conditions. Inanother embodiment, the degree of pH stability may be assayed bymeasuring the activity or specific activity of the enzyme in specific pHconditions. The specific pH conditions may be any pH from pH5 to pH10.5.

Thus, the PS4 variant polypeptide may have a longer half life, or ahigher activity (depending on the assay) when compared to the parentpolypeptide under identical conditions. The PS4 variant polypeptides mayhave 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or longerhalf life when compared to their parent polypeptides under identical pHconditions. Alternatively, or in addition, they may have such higheractivity when compared to the parent polypeptide under identical pHconditions.

Exo-Specificity

It is known that some non-maltogenic exoamylases can have some degree ofendoamylase activity. In some cases, this type of activity may need tobe reduced or eliminated since endoamylase activity can possiblynegatively effect the quality of the final bread product by producing asticky or gummy crumb due to the accumulation of branched dextrins.

We provide PS4 variant polypeptides which have a higher exo-epecificitycompared to the parent polypeptide or a wild type polypeptide whentested under the same conditions. Specifically, we provide for PS4variant polypeptides comprising mutations at any one or more ofpositions 26, 70, 121, 145, 161, 223, 223, 303, 306, 309, 339, 339, morepreferably any one or more of mutations 26E, 70D, 121A, 121 D, 121H,121M, 121W, 121Y, 145D, 161A, 223A, 223E, 223K, 223R, 223V, 303D, 303E,306G, 306T, 309P, 339A, 339E.

Exo-specificity can usefully be measured by determining the ratio oftotal amylase activity to the total endoamylase activity. This ratio isreferred to in this document as a “Exo-specificity index”. In preferredembodiments, an enzyme is considered an exoamylase if it has aexo-specificity index of 20 or more, i.e., its total amylase activity(including exo-amylase activity) is 20 times or more greater than itsendoamylase activity. In highly preferred embodiments, theexo-specificity index of exoamylases is 30 or more, 40 or more, 50 ormore, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more. Inhighly preferred embodiments, the exo-specificity index is 150 or more,200 or more, 300 or more, 400 or more, 500 or more or 600 or more.

The total amylase activity and the endoamylase activity may be measuredby any means known in the art. For example, the total amylase activitymay be measured by assaying the total number of reducing ends releasedfrom a starch substrate. Alternatively, the use of a Betamyl assay isdescribed in further detail in the Examples, and for convenience,amylase activity as assayed in the Examples is described in terms of“Betamyl Units” in the Tables.

Endoamylase activity may be assayed by use of a Phadebas Kit (Pharmaciaand Upjohn). This makes use of a blue labelled crosslinked starch(labelled with an azo dye); only internal cuts in the starch moleculerelease label, while external cuts do not do so. Release of dye may bemeasured by spectrophotometry. Accordingly, the Phadebas Kit measuresendoamylase activity, and for convenience, the results of such an assay(described in the Examples) are referred to in this document as“Phadebas units”.

In a highly preferred embodiment, therefore, the exo-specificity indexis expressed in terms of Betamyl Units/Phadebas Units, also referred toas “B/Phad”.

Exo-specificity may also be assayed according to the methods describedin the prior art, for example, in our International Patent PublicationNumber WO99/50399. This measures exo-specificity by way of a ratiobetween the endoamylase activity to the exoamylase activity. Thus, in apreferred aspect, the PS4 variants described here will have less than0.5 endoamylase units (EAU) per unit of exoamylase activity. Preferablythe non-maltogenic exoamylases which are suitable for use according tothe present invention have less than 0.05 EAU per unit of exoamylaseactivity and more preferably less than 0.01 EAU per unit of exoamylaseactivity.

The PS4 variants described here will preferably have exospecificity, forexample measured by exo-specificity indices, as described above,consistent with their being exoamylases. Moreover, they preferably havehigher or increased exospecificity when compared to the parent enzymesor polypeptides from which they are derived. Thus, for example, the PS4variant polypeptides may have 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200% or higher exo-specificity index when compared to theirparent polypeptides, preferably under identical conditions. They mayhave 1.5× or higher, 2× or higher, 5× or higher, 10× or higher, 50× orhigher, 100× or higher, when compared to their parent polypeptides,preferably under identical conditions.

Uses of PS4 Variant Polypeptides and Nucleic Acids

The PS4 variant polypeptides, nucleic acids, host cells, expressionvectors, etc, may be used in any application for which an amylase may beused. In particular, they may be used to substitute for anynon-maltogenic exoamylase. They may be used to supplement amylase ornon-maltogenic exoamylase activity, whether alone or in combination withother known amylases or non-maltogenic exoamylases.

The PS4 variant sequences described here may be used in variousapplications in the food industry—such as in bakery and drink products,they may also be used in other applications such as a pharmaceuticalcomposition, or even in the chemical industry. In particular, the PS4variant polypeptides and nucleic acids are useful for various industrialapplications including baking (as disclosed in WO 99/50399) and flourstandardisation (volume enhancement or improvement). They may be used toproduce maltotetraose from starch and other substrates.

We therefore describe a method for preparing a food product, the methodcomprising: (a) obtaining a non-maltogenic exoamylase; (b) introducing amutation at any one or more of the positions of the non-maltogenicexoamylase as set out in this document; (c) admixing the resultingpolypeptide with a food ingredient.

The PS4 variant polypeptides may be used to enhance the volume of bakeryproducts such as bread. While not wishing to be bound by any particulartheory, we believe that this results from the reduction in viscosity ofthe dough during heating (such as baking) as a result of the exoamylaseshortening amylose molecules. This enables the carbon dioxide generatedby fermentation to increase the size of the bread with less hindrance.

Thus, food products comprising or treated with PS4 variant polypeptidesare expanded in volume when compared to products which have not been sotreated, or treated with parent polypeptides. In other words, the foodproducts have a larger volume of air per volume of food product.Alternatively, or in addition, the food products treated with PS4variant polypeptides have a lower density, or weight (or mass) pervolume ratio. In particularly preferred embodiments, the PS4 variantpolypeptides are used to enhance the volume of bread. Volume enhancementor expansion is beneficial because it reduces the gumminess orstarchiness of foods. Light foods are preferred by consumers, and thecustomer experience is enhanced. In preferred embodiments, the use ofPS4 variant polypeptides enhances the volume by 10%, 20%, 30% 40%, 50%or more.

The use of PS4 variant polypeptides to increase the volume of foods isdescribed in detail in the Examples.

Food Uses

The PS4 variant polypeptides and nucleic acids described here may beused as—or in the preparation of—a food. In particular, they may beadded to a food, i.e., as a food additive. The term “food” is intendedto include both prepared food, as well as an ingredient for a food, suchas a flour. In a preferred aspect, the food is for human consumption.The food may be in the from of a solution or as a solid—depending on theuse and/or the mode of application and/or the mode of administration.

The PS4 variant polypeptides and nucleic acids may be used as a foodingredient. As used herein the term “food ingredient” includes aformulation, which is or can be added to functional foods or foodstuffsand includes formulations which can be used at low levels in a widevariety of products that require, for example, acidifying oremulsifying. The food ingredient may be in the from of a solution or asa solid—depending on the use and/or the mode of application and/or themode of administration.

The PS4 variant polypeptides and nucleic acids disclosed here may be—ormay be added to—food supplements. The PS4 variant polypeptides andnucleic acids disclosed here may be—or may be added to—functional foods.As used herein, the term “functional food” means food which is capableof providing not only a nutritional effect and/or a taste satisfaction,but is also capable of delivering a further beneficial effect toconsumer. Although there is no legal definition of a functional food,most of the parties with an interest in this area agree that they arefoods marketed as having specific health effects.

The PS4 variant polypeptides may also be used in the manufacture of afood product or a foodstuff. Typical foodstuffs include dairy products,meat products, poultry products, fish products and dough products. Thedough product may be any processed dough product, including fried, deepfried, roasted, baked, steamed and boiled doughs, such as steamed breadand rice cakes. In highly preferred embodiments, the food product is abakery product.

Preferably, the foodstuff is a bakery product. Typical bakery (baked)products include bread—such as loaves, rolls, buns, pizza bases etc.pastry, pretzels, tortillas, cakes, cookies, biscuits, krackers etc.

We therefore describe a method of modifying a food additive comprising anon-maltogenic exoamylase, the method comprising introducing a mutationat any one or more of the positions of the non-maltogenic exoamylase asset out in this document. The same method can be used to modify a foodingredient, or a food supplement, a food product, or a foodstuff.

Retrogradation/Staling

We describe the use of PS4 variant proteins that are capable ofretarding the staling of starch media, such as starch gels. The PS4variant polypeptides are especially capable of retarding the detrimentalretrogradation of starch.

Most starch granules are composed of a mixture of two polymers: anessentially linear amylose and a highly branched amylopectin.Amylopectin is a very large, branched molecule consisting of chains ofα-D-glucopyranosyl units joined by (1-4) linkages, wherein said chainsare attached by α-D-(1-6) linkages to form branches. Amylopectin ispresent in all natural starches, constituting about 75% of most commonstarches. Amylose is essentially a linear chain of (1-4) linkedα-D-glucopyranosyl units having few α-D-(1-6) branches. Most starchescontain about 25% amylose.

Starch granules heated in the presence of water undergo anorder-disorder phase transition called gelatinization, where liquid istaken up by the swelling granules. Gelatinization temperatures vary fordifferent starches. Upon cooling of freshly baked bread the amylosefraction, within hours, retrogrades to develop a network. This processis beneficial in that it creates a desirable crumb structure with a lowdegree of firmness and improved slicing properties. More graduallycrystallisation of amylopectin takes place within the gelatinised starchgranules during the days after baking. In this process amylopectin isbelieved to reinforce the amylose network in which the starch granulesare embedded. This reinforcement leads to increased firmness of thebread crumb. This reinforcement is one of the main causes of breadstaling.

It is known that the quality of baked products gradually deterioratesduring storage As a consequence of starch recystallisation (also calledretrogradation), the water-holding capacity of the crumb is changed withimportant implications on the organoleptic and dietary properties. Thecrumb loses softness and elasticity and becomes firm and crumbly. Theincrease in crumb firmness is often used as a measure of the stalingprocess of bread.

The rate of detrimental retrogradation of amylopectin depends on thelength of the side chains of amylopectin. Thus, enzymatic hydrolysis ofthe amylopectin side chains, for example, by PS4 variant polypeptideshaving non-maltogenic exoamylase activity, can markedly reduce theircrystallisation tendencies.

Accordingly, the use of PS4 variant polypeptides as described here whenadded to the starch at any stage of its processing into a food product,e.g., before during or after baking into bread can retard or impede orslow down the retrogradation. Such use is described in further detailbelow.

We therefore describe a method of improving the ability of anon-maltogenic exoamylase to prevent staling, preferably detrimentalretrogradation, of a dough product, the method comprising introducing amutation at any one or more of the positions of the non-maltogenicexoamylase as set out in this document.

Assays for Measurement of Retrogradation (Inc. Staling)

For evaluation of the antistaling effect of the PS4 variant polypeptideshaving non-maltogenic exoamylase activity described here, the crumbfirmness can be measured 1, 3 and 7 days after baking by means of anInstron 4301 Universal Food Texture Analyzer or similar equipment knownin the art.

Another method used traditionally in the art and which is used toevaluate the effect on starch retrogradation of a PS4 variantpolypeptide having non-maltogenic exoamylase activity is based on DSC(differential scanning calorimetry). Here, the melting enthalpy ofretrograded amylopectin in bread crumb or crumb from a model systemdough baked with or without enzymes (control) is measured. The DSCequipment applied in the described examples is a Mettler-Toledo DSC 820run with a temperature gradient of 10° C. per min from 20 to 95° C. Forpreparation of the samples 10-20 mg of crumb are weighed and transferredinto Mettler-Toledo aluminium pans which then are hermetically sealed.

The model system doughs used in the described examples contain standardwheat flour and optimal amounts of water or buffer with or without thenon-maltogenic PS4 variant exoamylase. They are mixed in a 10 or 50 gBrabender Farinograph for 6 or 7 min., respectively. Samples of thedoughs are placed in glass test tubes (15*0.8 cm) with a lid. These testtubes are subjected to a baking process in a water bath starting with 30min. incubation at 33° C. followed by heating from 33 to 95° C. with agradient of 1.1° C. per min. and finally a 5 min. incubation at 95° C.Subsequently, the tubes are stored in a thermostat at 20° C. prior toDSC analysis.

In preferred embodiments, the PS4 variants described here have a reducedmelting enthalpy, compared to the control. In highly preferredembodiments, the PS4 variants have a 10% or more reduced meltingenthalpy. Preferably, they have a 20% or more, 30%, 40%, 50%, 60%, 70%,80%, 90% or more reduced melting enthalpy when compared to the control.

TABLE 2 DSC (J/g) Control 2.29 0.5 D34 1.91   1 D34 1.54   2 D34 1.14

The above Table 2 shows DSC values of model dough systems prepared withdifferent doses of PSac-D34 after 7 days of storage. 0.5, 1 and 2 partsper million (or microgram per gram) of flour are tested.

Preparation of Starch Products

We provide the use of PS4 variant polypeptides in the preparation offood products, in particular, starch products. The method comprisesforming the starch product by adding a non-maltogenic exoamylase enzymesuch as a PS4 variant polypeptide, to a starch medium. If the starchmedium is a dough, then the dough is prepared by mixing together flour,water, the non-maltogenic exoamylase which is a PS4 variant polypeptideand optionally other possible ingredients and additives.

The term “starch” should be taken to mean starch per se or a componentthereof, especially amylopectin. The term “starch medium” means anysuitable medium comprising starch. The term “starch product” means anyproduct that contains or is based on or is derived from starch.Preferably, the starch product contains or is based on or is derivedfrom starch obtained from wheat flour. The term “flour” as used hereinis a synonym for the finely-ground meal of wheat or other grain.Preferably, however, the term means flour obtained from wheat per se andnot from another grain. Thus, and unless otherwise expressed, referencesto “wheat flour” as used herein preferably mean references to wheatflour per se as well as to wheat flour when present in a medium, such asa dough.

A preferred flour is wheat flour or rye flour or mixtures of wheat andrye flour. However, dough comprising flour derived from other types ofcereals such as for example from rice, maize, barley, and durra are alsocontemplated. Preferably, the starch product is a bakery product. Morepreferably, the starch product is a bread product. Even more preferably,the starch product is a baked farinaceous bread product. The term “bakedfarinaceous bread product” refers to any baked product based on a doughobtainable by mixing flour, water, and a leavening agent under doughforming conditions. Further components can of course be added to thedough mixture.

Thus, if the starch product is a baked farinaceous bread product, thenthe process comprises mixing—in any suitable order—flour, water, and aleavening agent under dough forming conditions and further adding a PS4variant polypeptide, optionally in the form of a premix. The leaveningagent may be a chemical leavening agent such as sodium bicarbonate orany strain of Saccharomyces cerevisiae (Baker's Yeast).

The PS4 variant non-maltogenic exoamylase can be added together with anydough ingredient including the water or dough ingredient mixture or withany additive or additive mixture. The dough can be prepared by anyconventional dough preparation method common in the baking industry orin any other industry making flour dough based products.

Baking of farinaceous bread products such as for example white bread,bread made from bolted rye flour and wheat flour, rolls and the like istypically accomplished by baking the bread dough at oven temperatures inthe range of from 180 to 250° C. for about 15 to 60 minutes. During thebaking process a steep temperature gradient (200→120° C.) is prevailingin the outer dough layers where the characteristic crust of the bakedproduct is developed. However, owing to heat consumption due to steamgeneration, the temperature in the crumb is only close to 100° C. at theend of the baking process.

We therefore describe a process for making a bread product comprising:(a) providing a starch medium; (b) adding to the starch medium a PS4variant polypeptide as described in this document; and (c) applying heatto the starch medium during or after step (b) to produce a breadproduct. We also describe a process for making a bread productcomprising adding to a starch medium a PS4 variant polypeptide asdescribed.

The non-maltogenic exoamylase PS4 variant polypeptide can be added as aliquid preparation or as a dry pulverulent composition either comprisingthe enzyme as the sole active component or in admixture with one or moreadditional dough ingredient or dough additive.

Improving Composition

We describe improver compositions, which include bread improvingcompositions and dough improving compositions. These comprise a PS4variant polypeptide, optionally together with a further ingredient, or afurther enzyme, or both.

We also provide for the use of such a bread and dough improvingcompositions in baling. In a further aspect, we provide a baked productor dough obtained from the bread improving composition or doughimproving composition. In another aspect, we describe a baked product ordough obtained from the use of a bread improving composition or a doughimproving composition.

Dough Preparation

A dough may be prepared by admixing flour, water, a dough improvingcomposition comprising PS4 variant polypeptide (as described above) andoptionally other ingredients and additives.

The dough improving composition can be added together with any doughingredient including the flour, water or optional other ingredients oradditives. The dough improving composition can be added before the flouror water or optional other ingredients and additives. The doughimproving composition can be added after the flour or water, or optionalother ingredients and additives. The dough can be prepared by anyconventional dough preparation method common in the baking industry orin any other industry making flour dough based products.

The dough improving composition can be added as a liquid preparation orin the form of a dry powder composition either comprising thecomposition as the sole active component or in admixture with one ormore other dough ingredients or additive.

The amount of the PS4 variant polypeptide non-maltogenic exoamylase thatis added is normally in an amount which results in the presence in thefinished dough of 50 to 100,000 units per kg of flour, preferably 100 to50,000 units per kg of flour. Preferably, the amount is in the range of200 to 20,000 units per kg of flour. Alternatively, the PS4 variantpolypeptide non-maltogenic exoamylase is added in an amount whichresults in the presence in the finished dough of 0.02-50 ppm of enzymebased on flour (0.02-50 mg enzyme per kg of flour), preferably 0.2-10ppm.

In the present context, 1 unit of the non-maltogenic exoamylase isdefined as the amount of enzyme which releases hydrolysis productsequivalent to 1 μmol of reducing sugar per min. when incubated at 50degrees C. in a test tube with 4 ml of 10 mg/ml waxy maize starch in 50mM MES, 2 mM calcium chloride, pH 6.0 as described hereinafter.

The dough as described here generally comprises wheat meal or wheatflour and/or other types of meal, flour or starch such as corn flour,corn starch, maize flour, rice flour, rye meal, rye flour, oat flour,oat meal, soy flour, sorghum meal, sorghum flour, potato meal, potatoflour or potato starch. The dough may be fresh, frozen, or part-baked.

The dough may be a leavened dough or a dough to be subjected toleavening. The dough may be leavened in various ways, such as by addingchemical leavening agents, e.g., sodium bicarbonate or by adding aleaven (fermenting dough), but it is preferred to leaven the dough byadding a suitable yeast culture, such as a culture of Saccharomycescerevisiae (baker's yeast), e.g. a commercially available strain of S.cerevisiae.

The dough may comprise fat such as granulated fat or shortening. Thedough may further comprise a further emulsifier such as mono- ordiglycerides, sugar esters of fatty acids, polyglycerol esters of fattyacids, lactic acid esters of monoglycerides, acetic acid esters ofmonoglycerides, polyoxethylene stearates, or lysolecithin.

We also describe a pre-mix comprising flour together with thecombination as described herein. The pre-mix may contain otherdough-improving and/or bread-improving additives, e.g. any of theadditives, including enzymes, mentioned herein.

Further Dough Additives or Ingredients

In order to improve further the properties of the baked product andimpart distinctive qualities to the baked product further doughingredients and/or dough additives may be incorporated into the dough.Typically, such further added components may include dough ingredientssuch as salt, grains, fats and oils, sugar or sweetener, dietary fibres,protein sources such as milk powder, gluten soy or eggs and doughadditives such as emulsifiers, other enzymes, hydrocolloids, flavouringagents, oxidising agents, minerals and vitamins

The emulsifiers are useful as dough strengtheners and crumb softeners.As dough strengtheners, the emulsifiers can provide tolerance withregard to resting time and tolerance to shock during the proofing.Furthermore, dough strengtheners will improve the tolerance of a givendough to variations in the fermentation time. Most dough strengthenersalso improve on the oven spring which means the increase in volume fromthe proofed to the baked goods. Lastly, dough strengtheners willemulsify any fats present in the recipe mixture.

Suitable emulsifiers include lecithin, polyoxyethylene stearat, mono-and diglycerides of edible fatty acids, acetic acid esters of mono- anddiglycerides of edible fatty acids, lactic acid esters of mono- anddiglycerides of edible fatty acids, citric acid esters of mono- anddiglycerides of edible fatty acids, diacetyl tartaric acid esters ofmono- and diglycerides of edible fatty acids, sucrose esters of ediblefatty acids, sodium stearoyl-2-lactylate, and calciumstearoyl-2-lactylate.

The further dough additive or ingredient can be added together with anydough ingredient including the flour, water or optional otheringredients or additives, or the dough improving composition. Thefurther dough additive or ingredient can be added before the flour,water, optional other ingredients and additives or the dough improvingcomposition. The further dough additive or ingredient can be added afterthe flour, water, optional other ingredients and additives or the doughimproving composition.

The further dough additive or ingredient may conveniently be a liquidpreparation. However, the further dough additive or ingredient may beconveniently in the form of a dry composition.

Preferably the further dough additive or ingredient is at least 1% theweight of the flour component of dough. More preferably, the furtherdough additive or ingredient is at least 2%, preferably at least 3%,preferably at least 4%, preferably at least 5%, preferably at least 6%.If the additive is a fat, then typically the fat may be present in anamount of from 1 to 5%, typically 1 to 3%, more typically about 2%.

Further Enzyme

In addition to the PS4 variant polypeptides, one or more further enzymesmay be used, for example added to the food, dough preparation, foodstuffor starch composition.

Further enzymes that may be added to the dough include oxidoreductases,hydrolases, such as lipases and esterases as well as glycosidases likeα-amylase, pullulanase, and xylanase. Oxidoreductases, such as forexample glucose oxidase and hexose oxidase, can be used for doughstrengthening and control of volume of the baked products and xylanasesand other hemicellulases may be added to improve dough handlingproperties, crumb softness and bread volume. Lipases are useful as doughstrengtheners and crumb softeners and α-amylases and other amylolyticenzymes may be incorporated into the dough to control bread volume andfurther reduce crumb firmness.

Further enzymes that may be used may be selected from the groupconsisting of a cellulase, a hemicellulase, a starch degrading enzyme, aprotease, a lipoxygenase.

Examples of useful oxidoreductases include oxidises such as maltoseoxidising enzyme, a glucose oxidase (EC 1.1.3.4), carbohydrate oxidase,glycerol oxidase, pyranose oxidase, galactose oxidase (EC 1.1.3.10) andhexose oxidase (EC 1.1.3.5).

Among starch degrading enzymes, amylases are particularly useful asdough improving additives. α-amylase breaks downs starch into dextrinswhich are further broken down by β-amylase to maltose. Other usefulstarch degrading enzymes which may be added to a dough compositioninclude glucoamylases and pullulanases.

Preferably, the further enzyme is at least a xylanase and/or at least anamylase. The term “xylanase” as used herein refers to xylanases (EC3.2.1.32) which hydrolyse xylosidic linkages.

The term “amylase” as used herein refers to amylases such as α-amylases(EC 3.2.1.1), β-amylases (EC 3.2.1.2) and γ-amylases (EC 3.2.1.3.

The further enzyme can be added together with any dough ingredientincluding the flour, water or optional other ingredients or additives,or the dough improving composition. The further enzyme can be addedbefore the flour, water, and optionally other ingredients and additivesor the dough improving composition. The further enzyme can be addedafter the flour, water, and optionally other ingredients and additivesor the dough improving composition. The further enzyme may convenientlybe a liquid preparation. However, the composition may be conveniently inthe form of a dry composition.

Some enzymes of the dough improving composition are capable ofinteracting with each other under the dough conditions to an extentwhere the effect on improvement of the theological and/or machineabilityproperties of a flour dough and/or the quality of the product made fromdough by the enzymes is not only additive, but the effect issynergistic.

In relation to improvement of the product made from dough (finishedproduct), it may be found that the combination results in a substantialsynergistic effect in respect to crumb structure. Also, with respect tothe specific volume of baked product a synergistic effect may be found.

The further enzyme may be a lipase (EC 3.1.1) capable of hydrolysingcarboxylic ester bonds to release carboxylate. Examples of lipasesinclude but are not limited to triacylglycerol lipase (EC 3.1.1.3),galactolipase (EC 3.1.1.26), phospholipase A1 (EC 3.1.1.32,phospholipase A2 (EC 3.1.1.4) and lipoprotein lipase A2 (EC 3.1.1.34).

Other Uses

The PS4 variants are suitable for the production of maltose and highmaltose syrups. Such products are of considerable interest in theproduction of certain confectioneries because of the low hygroscoposity,low viscosity, good heat stability and mild, not too sweet taste ofmaltose. The industrial process of producing maltose syrups comprisesliquefying starch, then saccharification with a maltose producingenzyme, and optionally with an enzyme cleaving the 1.6-branching pointsin amylopectin, for instance an .alpha-1,6-amyloglucosidase.

The PS4 variants described here may be added to and thus become acomponent of a detergent composition. The detergent composition may forexample be formulated as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations. In a specific aspect, we describe a detergentadditive comprising the PS4 variant. The detergent additive as well asthe detergent composition may comprise one or more other enzymes such asa protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase. In generalthe properties of the chosen enzyme(s) should be compatible with theselected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

The PS4 variant may also be used in the production of lignocellulosicmaterials, such as pulp, paper and cardboard, from starch reinforcedwaste paper and cardboard, especially where repulping occurs at pH above7 and where amylases can facilitate the disintegration of the wastematerial through degradation of the reinforcing starch. The PS4 variantsmay especially be useful in a process for producing a papermaking pulpfrom starch-coated printed paper. The process may be performed asdescribed in WO 95/14807, comprising the following steps: a)disintegrating the paper to produce a pulp, b) treating with astarch-degrading enzyme before, during or after step a), and c)separating ink particles from the pulp after steps a) and b). The PS4variant may also be very useful in modifying starch where enzymaticallymodified starch is used in papermaking together with alkaline fillerssuch as calcium carbonate, kaolin and clays. With the PS4 variantsdescribed here it becomes possible to modify the starch in the presenceof the filler thus allowing for a simpler integrated process. A PS4variant may also be very useful in textile desizing. In the textileprocessing industry, amylases are traditionally used as auxiliaries inthe desizing process to facilitate the removal of starch-containing sizewhich has served as a protective coating on weft yarns during weaving.Complete removal of the size coating after weaving is import-ant toensure optimum results in the subsequent processes, in which the fabricis scoured, bleached and dyed. Enzymatic starch break-down is preferredbecause it does not involve any harmful effect on the fiber material.The PS4 variant may be used alone or in combination with a cellulasewhen desizing cellulose-containing fabric or textile.

The PS4 variant may also be an amylase of choice for production ofsweeteners from starch A “traditional” process for conversion of starchto fructose syrups normally consists of three consecutive enzymaticprocesses, viz., a liquefaction process followed by a saccharificationprocess and an isomerization process. During the liquefaction process,starch is degraded to dextrins by an amylase at pH values between 5.5and 6.2 and at temperatures of 95-160° C. for a period of approx. 2hours. In order to ensure an optimal enzyme stability under theseconditions, 1 mM of calcium is added (40 ppm free calcium ions). Afterthe liquefaction process the dextrins are converted into dextrose byaddition of a glucoamylase and a debranching enzyme, such as anisoamylase or a pullulanase. Before this step the pH is reduced to avalue below 4.5, maintaining the high temperature (above 95° C.), andthe liquefying amylase activity is denatured. The temperature is loweredto 60° C., and glucoamylase and debranching enzyme are added. Thesaccharification process proceeds for 24-72 hours.

Reed Applications

In one embodiment, the PS4 variant polypeptide is capable of degradingresistant starch.

As used herein the term ‘degrading’ relates to the partial or completehydrolysis or degradation of resistant starch to glucose and/oroligosaccharides—such as maltose and/or dextrins.

The PS4 variant polypeptide may degrade residual resistant starch thathas not been completely degraded by an animals amylase. By way ofexample, the PS4 variant polypeptide may be used to assist an animal'samylase (e.g. pancreatic amylase) in improving the degradation ofresistant starch. Pancreatic α-amylase is excreted in the digestivesystem by animals. Pancreatic α-amylase degrades starch in the feed.However, a part of the starch, the resistant starch, is not degradedfully by the pancreatic α-amylase and is therefore not absorbed in thesmall intestine (see definition of resistant starch). The PS4 variantpolypeptide in some embodiments is able to assist the pancreaticα-amylase in degrading starch in the digestive system and therebyincrease the utilisation of starch by the animal.

The ability of an enzyme to degrade resistant-starch may be analysed forexample by a method developed and disclosed by Megazyme InternationalIreland Ltd. for the measurement of resistant starch, solubilised starchand total starch content of a sample (Resistant Starch Assay Procedure,AOAC Method 2002.02, AACC Method 32-40).

Accordingly, the PS4 variant polypeptides may be ingested by an animalfor beneficial purposes, and may therefore be incorporated into animalfeeds.

We therefore disclose the use of a PS4 variant polypeptide as acomponent for use in a feed comprising starch, or for use in a feedimprovement composition, in which the PS4 variant polypeptide is capableof degrading resistant starch. We also disclose a feed comprising astarch and a PS4 variant polypeptide. We further disclose a method ofdegrading resistant starch in a feed comprising contacting saidresistant starch with a PS4 variant polypeptide.

We further describe the use of a PS4 variant polypeptide in thepreparation of a feed comprising a starch, to degrade resistant starch.Furthermore, we disclose the use of a PS4 variant polypeptide in thepreparation of a feed to improve the calorific value of said feed. Wedisclose the use of an enzyme in the preparation of a feed to improveanimal performance. In a further embodiment, we describe a process forpreparing a feed comprising admixing a starch and a PS4 variantpolypeptide enzyme.

By way of example, use of a component comprising PS4 variantpolypeptides and which is capable of degrading resistant starch isadvantageous because there is a marked increase in the degradation ofstarch and/or starch degradation products in an animal. Furthermore,such use is advantageous because there is a marked increase in thedigestibility of starch and/or starch degradation products by an animal.Furthermore, such use is advantageous because it provides a means ofenhancing the efficiency of deriving energy from a feed by an animal.Furthermore, such use is advantageous because it provides a means toenhance the bioavailability of resistant starch.

Animal Feeds

Animal feeds for which the PS4 variant polypeptides are suitable for usemay be formulated to meet the specific needs of particular animal groupsand to provide the necessary carbohydrate, fat, protein and othernutrients in a form that can be metabolised by the animal.

Preferably, the animal feed is a feed for swine or poultry.

As used herein the term ‘swine’ relates to non-ruminant omnivores suchas pigs, hogs or boars. Typically, swine feed includes about 50 percentcarbohydrate, about 20 percent protein and about 5% fat. An example of ahigh energy swine feed is based on corn which is often combined withfeed supplements for example, protein, minerals, vitamins and aminoacids such as lysine and tryptophan. Examples of swine feeds includeanimal protein products, marine products, milk products, grain productsand plant protein products, all of which may further comprise naturalflavourings, artificial flavourings, micro and macro minerals, animalfats, vegetable fats, vitamins, preservatives or medications such asantibiotics.

It is to be understood that where reference is made in the presentspecification, including the accompanying claims, to ‘swine feed’ suchreference is meant to include “transition” or “starter” feeds (used towean young swine) and “finishing” or “grower” feeds (used following thetransition stage for growth of swine to an age and/or size suitable formarket).

As used herein the term ‘poultry’ relates to fowl such as chickens,broilers, hens, roosters, capons, turkeys, ducks, game fowl, pullets orchicks. Poultry feeds may be referred to as “complete” feeds becausethey contain all the protein, energy, vitamin, minerals, and othernutrients necessary for proper growth, egg production, and health of thebirds. However, poultry feeds may further comprise vitamins, minerals ormedications such as coccidiostats (for example Monensin sodium,Lasalocid, Amprolium, Salinomycin, and Sulfaquinoxaline) and/orantibiotics (for example Penicillin, Bacitracin, Chlortetracycline, andOxytetracycline).

Young chickens or broilers, turkeys and ducks kept for meat productionare fed differently from pullets saved for egg production. Broilers,ducks and turkeys have larger bodies and gain weight more rapidly thando the egg-producing types of chickens. Therefore, these birds are feddiets with higher protein and energy levels.

It is to be understood that where reference is made in the presentspecification, including the accompanying claims, to ‘poultry feed’ suchreference is meant to include “starter” feeds (post-hatching),“finisher”, “grower” or “developer” feeds (from 6-8 weeks of age untilslaughter size reached) and “layer” feeds (fed during egg production).

Animal feeds may be formulated to meet the animal's nutritional needswith respect to, for example, meat production, milk production, eggproduction, reproduction and response to stress. In addition, the animalfeeds are formulated to improve manure quality.

In a preferred aspect the animal feed contains a raw material such as alegume, for example pea or soy or a cereal, for example wheat, corn(maize), rye or barley. Suitably, the raw material may be potato.

Feed Stuffs

The PS4 variant polypeptides may be used in feeds for animal consumptionby the indirect or direct application of the PS4 variant polypeptides tothe feed, whether alone or in combination with other ingredients, suchas food ingredients.

Typical food ingredients may include any one or more of an additive suchas an animal or vegetable fat, a natural or synthetic seasoning,antioxidant, viscosity modifier, essential oil, and/or flavour, dyeand/or colorant, vitamin, mineral, natural and/or non-natural aminoacid, nutrient, additional enzyme (including genetically manipulatedenzymes), a binding agent such as guar gum or xanthum gum, buffer,emulsifier, lubricant, adjuvant, suspending agent, preservative, coatingagent or solubilizing agent and the like.

Examples of the application methods include, but are not limited to,coating the feed in a material comprising the PS4 variant polypeptide,direct application by mixing the PS4 variant polypeptide with the feed,spraying the PS4 variant polypeptide onto the feed surface or dippingthe feed into a preparation of the PS4 variant polypeptide.

The PS4 variant polypeptide is preferably applied by mixing it with afeed or by spraying onto feed particles for animal consumption.Alternatively, the PS4 variant polypeptide may be included in theemulsion of a feed, or the interior of solid products by injection ortumbling.

The PS4 variant polypeptide may be applied to intersperse, coat and/orimpregnate a feed. Mixtures with other ingredients may also be used andmay be applied separately, simultaneously or sequentially. Chelatingagents, binding agents, emulsifiers and other additives such as microand macro minerals, amino acids, vitamins, animal fats, vegetable fats,preservatives, flavourings, colourings, may be similarly applied to thefeed simultaneously (either in mixture or separately) or appliedsequentially.

Amount of PS4 Variant Polypeptide

The optimum amount of the PS4 variant polypeptide to be used will dependon the feed to be treated and/or the method of contacting the feed withthe PS4 variant polypeptide and/or the intended use for the same. Theamount of PS4 variant polypeptide should be in a sufficient amount to beeffective to substantially degrade resistant starch following ingestionand during digestion of the feed.

Advantageously, the PS4 variant polypeptide would remain effectivefollowing ingestion of a feed for animal consumption and duringdigestion of the feed until a more complete digestion of the feed isobtained, i.e. an increased calorific value of the feed is released.

Amylase Combinations

We disclose in particular combinations of PS4 variant polypeptides withamylases, in particular, maltogenic amylases. Maltogenic alpha-amylase(glucan 1,4-a-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyzeamylose and amylopectin to maltose in the alpha-configuration.

A maltogenic alpha-amylase from Bacillus (EP 120 693) is commerciallyavailable under the trade name Novamyl (Novo Nordisk A/S, Denmark) andis widely used in the baking industry as an anti-staling agent due toits ability to reduce retrogradation of starch Novamyl is described indetail in International Patent Publication WO 91/04669. The maltogenicalpha-amylase Novamyl shares several characteristics with cyclodextringlucanotransferases (CGTases), including sequence homology (Henrissat B,Bairoch A; Biochem J., 316, 695-696 (1996)) and formation oftransglycosylation products (Christophersen, C., et al., 1997, Starch,vol. 50, No. 1, 39-45).

In highly preferred embodiments, we disclose combinations comprising PS4variant polypeptides together with Novamyl or any of its variants. Suchcombinations are useful for food production such as baling. The Novamylmay in particular comprise Novamyl 1500 MG.

Other documents describing Novamyl and its uses include Christophersen,C., Pedersen, S., and Christensen, T., (1993) Method for production ofmaltose an a limit dextrin, the limit dextrin, and use of the limitdextrin. Denmark, and WO 95/10627. It is further described in U.S. Pat.No. 4,598,048 and U.S. Pat. No. 4,604,355. Each of these documents ishereby incorporated by reference, and any of the Novamyl polypeptidesdescribed therein may be used in combinations with any of the PS4variant polypeptides described here.

Variants, homologues, and mutants of Novamyl may be used for thecombinations, provided they retain alpha amylase activity. For example,any of the Novamyl variants disclosed in U.S. Pat. No. 6,162,628, theentire disclosure of which is hereby incorporated by reference, may beused in combination with the PS4 variant polypeptides described here. Inparticular, any of the polypeptides described in that document,specifically variants of SEQ ID NO: 1 of U.S. Pat. No. 6,162,628 at anyone or more positions corresponding to Q13, I16, D17, N26, N28, P29,A30, S32, Y33, G34, L35, K40, M45, P73, V74, D76 N77, D79, N86, R95,N99, I100, H103, Q119, N120, N131, S141, T142, A148, N152, A163, H169,N171, G172, I174, N176, N187, F188, A192, Q201, N203, H220, N234, G236,Q247, K249, D261, N266, L268, R272, N275, N276, V279, N280, V281, D285,N287, F297, Q299, N305, K316, N320, L321, N327, A341, N342, A348, Q365,N371, N375, M78, G397, A381, F389, N401, A403, K425, N436, S442, N454,N468, N474, S479, A483, A486, V487, S493, T494, S495, A496, S497, A498,Q500, N507, 1510, N513, K520, Q526, A555, A564, S573, N575, Q581, S583,F586, K589, N595, G618, N621, Q624, A629, F636, K645, N664 and/or T681may be used.

Amino Acid Sequences

The invention makes use of a PS4 variant nucleic acid, and the aminoacid sequences of such PS4 variant nucleic acids are encompassed by themethods and compositions described here.

As used herein, the term “amino acid sequence” is synonymous with theterm “polypeptide” and/or the term “protein”. In some instances, theterm “amino acid sequence” is synonymous with the term “peptide”. Insome instances, the term “amino acid sequence” is synonymous with theterm “enzyme”.

The amino acid sequence may be prepared/isolated from a suitable source,or it may be made synthetically or it may be prepared by use ofrecombinant DNA techniques.

The PS4 variant enzyme described here may be used in conjunction withother enzymes. Thus we further disclose a combination of enzymes whereinthe combination comprises a PS4 variant polypeptide enzyme describedhere and another enzyme, which itself may be another PS4 variantpolypeptide enzyme.

PS4 Variant Nucleotide Sequence

As noted above, we disclose nucleotide sequences encoding the PS4variant enzymes having the specific properties described.

The term “nucleotide sequence” or “nucleic acid sequence” as used hereinrefers to an oligonucleotide sequence or polynucleotide sequence, andvariant, homologues, fragments and derivatives thereof (such as portionsthereof). The nucleotide sequence may be of genomic or synthetic orrecombinant origin, which may be double-stranded or single-strandedwhether representing the sense or anti-sense strand.

The term “nucleotide sequence” as used in this document includes genomicDNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, morepreferably cDNA sequence coding for a PS4 variant polypeptide.

Typically, the PS4 variant nucleotide sequence is prepared usingrecombinant DNA techniques (i.e. recombinant DNA). However, in analternative embodiment, the nucleotide sequence could be synthesised, inwhole or in part, using chemical methods well known in the art (seeCaruthers M H et al, (1980) Nuc Acids Res Symp Ser 215-23 and Horn T etal., (1980) Nuc Acids Res Symp Ser 225-232).

Preparation of Nucleic Acid Sequences

A nucleotide sequence encoding either an enzyme which has the specificproperties as defined herein (e.g., a PS4 variant polypeptide) or anenzyme which is suitable for modification, such as a parent enzyme, maybe identified and/or isolated and/or purified from any cell or organismproducing said enzyme. Various methods are well known within the art forthe identification and/or isolation and/or purification of nucleotidesequences. By way of example, PCR amplification techniques to preparemore of a sequence may be used once a suitable sequence has beenidentified and/or isolated and/or purified.

By way of further example, a genomic DNA and/or cDNA library may beconstructed using chromosomal DNA or messenger RNA from the organismproducing the enzyme. If the amino acid sequence of the enzyme or a partof the amino acid sequence of the enzyme is known, labelledoligonucleotide probes may be synthesised and used to identifyenzyme-encoding clones from the genomic library prepared from theorganism. Alternatively, a labelled oligonucleotide probe containingsequences homologous to another known enzyme gene could be used toidentify enzyme-encoding clones. In the latter case, hybridisation andwashing conditions of lower stringency are used.

Alternatively, enzyme-encoding clones could be identified by insertingfragments of genomic DNA into an expression vector, such as a plasmid,transforming enzyme-negative bacteria with the resulting genomic DNAlibrary, and then plating the transformed bacteria onto agar platescontaining a substrate for enzyme (i.e. maltose), thereby allowingclones expressing the enzyme to be identified.

In a yet further alternative, the nucleotide sequence encoding theenzyme may be prepared synthetically by established standard methods,e.g. the phosphoroamidite method described by Beucage S. L. et al.,(1981) Tetrahedron Letters 22, p 1859-1869, or the method described byMatthes et al., (1984) EMBO J. 3, p 801-805. In the phosphoroamiditemethod, oligonucleotides are synthesised, e.g. in an automatic DNAsynthesiser, purified, annealed, ligated and cloned in appropriatevectors.

The nucleotide sequence may be of mixed genomic and synthetic origin,mixed synthetic and cDNA origin, or mixed genomic and cDNA origin,prepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate) in accordance with standard techniques. Each ligatedfragment corresponds to various parts of the entire nucleotide sequence.The DNA sequence may also be prepared by polymerase chain reaction (PCR)using specific primers, for instance as described in U.S. Pat. No.4,683,202 or in Saiki R K et al., (Science (1988) 239, pp 487-491).

Variants/Homologues/Derivatives

We further describe the use of variants, homologues and derivatives ofany amino acid sequence of an enzyme or of any nucleotide sequenceencoding such an enzyme, such as a PS4 variant polypeptide or a PS4variant nucleic acid. Unless the context dictates otherwise, the term“PS4 variant nucleic acid” should be taken to include each of thenucleic acid entities described below, and the term “PS4 variantpolypeptide” should likewise be taken to include each of the polypeptideor amino acid entities described below.

Here, the term “homologue” means an entity having a certain homologywith the subject amino acid sequences and the subject nucleotidesequences. Here, the term “homology” can be equated with “identity”.

In the present context, a homologous sequence is taken to include anamino acid sequence which may be at least 75, 80, 85 or 90% identical,preferably at least 95, 96, 97, 98 or 99% identical to the subjectsequence. Typically, the homologues will comprise the same active sitesetc. as the subject amino acid sequence. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of this documentit is preferred to express homology in terms of sequence identity.

In the present context, an homologous sequence is taken to include anucleotide sequence which may be at least 75, 80, 85 or 90% identical,preferably at least 95, 96, 97, 98 or 99% identical to a nucleotidesequence encoding a PS4 variant polypeptide enzyme (such as a PS4variant nucleic acid). Typically, the homologues will comprise the samesequences that code for the active sites etc as the subject sequence.Although homology can also be considered in terms of similarity (i.e.amino acid residues having similar chemical properties/functions), inthe context of this document it is preferred to express homology interms of sequence identity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage the default gap penalty for amino acid sequences is −12 for agap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al 1984 Nuc.Acids Research 12 p 387). Examples of other software than can performsequence comparisons include, but are not limited to, the BLAST package(see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4^(th)Ed—Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) andthe GENEWORKS suite of comparison tools. Both BLAST and FASTA areavailable for offline and online searching (see Ausubel et al., 1999,Short Protocols in Molecular Biology, pages 7-58 to 760).

However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 andtatiana@ncbi.nlm.nih.gov).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). For some applications, it ispreferred to use the public default values for the GCG package, or inthe case of other software, the default matrix, such as BLOSUM62.

Alternatively, percentage homologies may be calculated using themultiple alignment feature in DNASIS™ (Hitachi Software), based on analgorithm, analogous to CLUSTAL (Higgins D G & Sharp P M (1988), Gene73(1), 237-244).

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in amino acid properties (such aspolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues) and it is therefore useful to groupamino acids together in functional groups. Amino acids can be groupedtogether based on the properties of their side chain alone. However itis more useful to include mutation data as well. The sets of amino acidsthus derived are likely to be conserved for structural reasons. Thesesets can be described in the form of a Venn diagram (Livingstone C. D.and Barton G. J. (1993) “Protein sequence alignments: a strategy for thehierarchical analysis of residue conservation” Comput. Appl. Biosci. 9:745-756)(Taylor W. R. (1986) “The classification of amino acidconservation” J. Theor. Biol. 119; 205-218). Conservative substitutionsmay be made, for example according to the table below which describes agenerally accepted Venn diagram grouping of amino acids.

Set Sub-set Hydro- F W Y H K M I L V A G C Aromatic F W Y H phobicAliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E DPositive- H K R ly charged Negative- E D ly charged SmallV C A G S P T N D Tiny A G S

We further disclose sequences comprising homologous substitution(substitution and replacement are both used herein to mean theinterchange of an existing amino acid residue, with an alternativeresidue) that may occur i.e. like-for-like substitution such as basicfor basic, acidic for acidic, polar for polar etc. Non-homologoussubstitution may also occur i.e. from one class of residue to another oralternatively involving the inclusion of unnatural amino acids such asornithine (hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine.

Variant amino acid sequences may include suitable spacer groups that maybe inserted between any two amino acid residues of the sequenceincluding alkyl groups such as methyl, ethyl or propyl groups inaddition to amino acid spacers such as glycine or β-alanine residues. Afurther form of variation, involves the presence of one or more aminoacid residues in peptoid form, will be well understood by those skilledin the art. For the avoidance of doubt, “the peptoid form” is used torefer to variant amino acid residues wherein the α-carbon substituentgroup is on the residue's nitrogen atom rather than the α-carbon.Processes for preparing peptides in the peptoid form are known in theart, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 andHorwell D C, Trends Biotechnol. (1995) 13(4), 132-134.

The nucleotide sequences described here, and suitable for use in themethods and compositions described here (such as PS4 variant nucleicacids) may include within them synthetic or modified nucleotides. Anumber of different types of modification to oligonucleotides are knownin the art. These include methylphosphonate and phosphorothioatebackbones and/or the addition of acridine or polylysine chains at the 3′and/or 5′ ends of the molecule. For the purposes of this document, it isto be understood that the nucleotide sequences described herein may bemodified by any method available in the art. Such modifications may becarried out in order to enhance the in vivo activity or life span ofnucleotide sequences.

We further describe the use of nucleotide sequences that arecomplementary to the sequences presented herein, or any derivative,fragment or derivative thereof. If the sequence is complementary to afragment thereof then that sequence can be used as a probe to identifysimilar coding sequences in other organisms etc.

Polynucleotides which are not 100% homologous to the PS4 variantsequences may be obtained in a number of ways. Other variants of thesequences described herein may be obtained for example by probing DNAlibraries made from a range of individuals, for example individuals fromdifferent populations. In addition, other homologues may be obtained andsuch homologues and fragments thereof in general will be capable ofselectively hybridising to the sequences shown in the sequence listingherein. Such sequences may be obtained by probing cDNA libraries madefrom or genomic DNA libraries from other species, and probing suchlibraries with probes comprising all or part of any one of the sequencesin the attached sequence listings under conditions of medium to highstringency. Similar considerations apply to obtaining species homologuesand allelic variants of the polypeptide or nucleotide sequencesdescribed here.

Variants and stain/species homologues may also be obtained usingdegenerate PCR which will use primers designed to target sequenceswithin the variants and homologues encoding conserved amino acidsequences. Conserved sequences can be predicted, for example, byaligning the amino acid sequences from several variants/homologues.Sequence alignments can be performed using computer software known inthe art. For example the GCG Wisconsin PileUp program is widely used.

The primers used in degenerate PCR will contain one or more degeneratepositions and will be used at stringency conditions lower than thoseused for cloning sequences with single sequence primers against knownsequences.

Alternatively, such polynucleotides may be obtained by site directedmutagenesis of characterised sequences. This may be useful where forexample silent codon sequence changes are required to optimise codonpreferences for a particular host cell in which the polynucleotidesequences are being expressed. Other sequence changes may be desired inorder to introduce restriction enzyme recognition sites, or to alter theproperty or function of the polypeptides encoded by the polynucleotides.

The polynucleotides (nucleotide sequences) such as the PS4 variantnucleic acids described in this document may be used to produce aprimer, e.g. a PCR primer, a primer for an alternative amplificationreaction, a probe e.g., labelled with a revealing label by conventionalmeans using radioactive or non-radioactive labels, or thepolynucleotides may be cloned into vectors. Such primers, probes andother fragments will be at least 15, preferably at least 20, for exampleat least 25, 30 or 40 nucleotides in length, and are also encompassed bythe term polynucleotides.

Polynucleotides such as DNA polynucleotides and probes may be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques. Ingeneral, primers will be produced by synthetic means, involving astepwise manufacture of the desired nucleic acid sequence one nucleotideat a time. Techniques for accomplishing this using automated techniquesare readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using a PCR (polymerase chain reaction) cloningtechniques. The primers may be designed to contain suitable restrictionenzyme recognition sites so that the amplified DNA can be cloned into asuitable cloning vector. Preferably, the variant sequences etc. are atleast as biologically active as the sequences presented herein.

As used herein “biologically active” refers to a sequence having asimilar structural function (but not necessarily to the same degree),and/or similar regulatory function (but not necessarily to the samedegree), and/or similar biochemical function (but not necessarily to thesame degree) of the naturally occurring sequence.

Hybridisation

We further describe sequences that are complementary to the nucleic acidsequences of PS4 variants or sequences that are capable of hybridisingeither to the PS4 variant sequences or to sequences that arecomplementary thereto.

The term “hybridisation” as used herein shall include “the process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing” as well as the process of amplification as carried out inpolymerase chain reaction (PCR) technologies. Therefore, we disclose theuse of nucleotide sequences that are capable of hybridising to thesequences that are complementary to the sequences presented herein, orany derivative, fragment or derivative thereof.

The term “variant” also encompasses sequences that are complementary tosequences that are capable of hybridising to the nucleotide sequencespresented herein.

Preferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising understringent conditions (e.g. 50° C. and 0.2×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein. Morepreferably, the term “variant” encompasses sequences that arecomplementary to sequences that are capable of hybridising under highstringent conditions (e.g. 65° C. and 0.1×SSC {1×SSC=0.15 M NaCl, 0.015M Na₃citrate pH 7.0}) to the nucleotide sequences presented herein.

We further disclose nucleotide sequences that can hybridise to thenucleotide sequences of PS4 variants (including complementary sequencesof those presented herein), as well as nucleotide sequences that arecomplementary to sequences that can hybridise to the nucleotidesequences of PS4 variants (including complementary sequences of thosepresented herein). We further describe polynucleotide sequences that arecapable of hybridising to the nucleotide sequences presented hereinunder conditions of intermediate to maximal stringency.

In a preferred aspect, we disclose nucleotide sequences that canhybridise to the nucleotide sequence of a PS4 variant nucleic acid, orthe complement thereof, under stringent conditions (e.g. 50° C. and0.2×SSC). More preferably, the nucleotide sequences can hybridise to thenucleotide sequence of a PS4 variant, or the complement thereof, underhigh stringent conditions (e.g. 65° C. and 0.1×SSC).

Site-Directed Mutagenesis

Once an enzyme-encoding nucleotide sequence has been isolated, or aputative enzyme-encoding nucleotide sequence has been identified, it maybe desirable to mutate the sequence in order to prepare an enzyme.Accordingly, a PS4 variant sequence may be prepared from a parentsequence. Mutations may be introduced using synthetic oligonucleotides.These oligonucleotides contain nucleotide sequences flanking the desiredmutation sites.

A suitable method is disclosed in Morinaga et al., (Biotechnology (1984)2, p 646-649). Another method of introducing mutations intoenzyme-encoding nucleotide sequences is described in Nelson and Long(Analytical Biochemistry (1989), 180, p 147-151). A further method isdescribed in Sarkar and Sommer (Biotechniques (1990), 8, p 404-407—“Themegaprimer method of site directed mutagenesis”).

In one aspect the sequence for use in the methods and compositionsdescribed here is a recombinant sequence—i.e. a sequence that has beenprepared using recombinant DNA techniques. These recombinant DNAtechniques are within the capabilities of a person of ordinary skill inthe art. Such techniques are explained in the literature, for example,J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Books 1-3, Cold Spring HarborLaboratory Press.

In one aspect the sequence for use in the methods and compositionsdescribed here is a synthetic sequence—i.e. a sequence that has beenprepared by in vitro chemical or enzymatic synthesis. It includes, butis not limited to, sequences made with optimal codon usage for hostorganisms—such as the methylotrophic yeasts Pichia and Hansenula.

The nucleotide sequence for use in the methods and compositionsdescribed here may be incorporated into a recombinant replicable vector.The vector may be used to replicate and express the nucleotide sequence,in enzyme form, in and/or from a compatible host cell. Expression may becontrolled using control sequences e.g. regulatory sequences. The enzymeproduced by a host recombinant cell by expression of the nucleotidesequence may be secreted or may be contained intracellularly dependingon the sequence and/or the vector used. The coding sequences may bedesigned with signal sequences which direct secretion of the substancecoding sequences through a particular prokaryotic or eukaryotic cellmembrane.

Expression of PS4 Nucleic Acids and Polypeptides

The PS4 polynucleotides and nucleic acids may include DNA and RNA ofboth synthetic and natural origin which DNA or RNA may contain modifiedor unmodified deoxy- or dideoxy-nucleotides or ribonucleotides oranalogs thereof. The PS4 nucleic acid may exist as single- ordouble-stranded DNA or RNA, an RNA/DNA heteroduplex or an RNA/DNAcopolymer, wherein the term “copolymer” refers to a single nucleic acidstrand that comprises both ribonucleotides and deoxyribonucleotides. ThePS4 nucleic acid may even be codon optimised to further increaseexpression.

The term “synthetic”, as used herein, is defined as that which isproduced by in vitro chemical or enzymatic synthesis. It includes but isnot limited to PS4 nucleic acids made with optimal codon usage for hostorganisms such as the methylotrophic yeasts Pichia and Hansenula.

Polynucleotides, for example variant PS4 polynucleotides described here,can be incorporated into a recombinant replicable vector. The vector maybe used to replicate the nucleic acid in a compatible host cell. Thevector comprising the polynucleotide sequence may be transformed into asuitable host cell. Suitable hosts may include bacterial, yeast, insectand fungal cells.

The term “transformed cell” includes cells that have been transformed byuse of recombinant DNA techniques. The transformation typically occursby insertion of one or more nucleotide sequences into a cell that is tobe transformed. The inserted nucleotide sequence may be a heterologousnucleotide sequence (i.e. is a sequence that is not natural to the cellthat is to be transformed. In addition, or in the alternative, theinserted nucleotide sequence may be an homologous nucleotide sequence(i.e. is a sequence that is natural to the cell that is to betransformed)—so that the cell receives one or more extra copies of anucleotide sequence already present in it.

Thus in a further embodiment, we provide a method of making PS4 variantpolypeptides and polynucleotides by introducing a polynucleotide into areplicable vector, introducing the vector into a compatible host cell,and growing the host cell under conditions which bring about replicationof the vector. The vector may be recovered from the host cell.

Expression Constructs

The PS4 nucleic acid may be operatively linked to transcriptional andtranslational regulatory elements active in a host cell of interest. ThePS4 nucleic acid may also encode a fusion protein comprising signalsequences such as, for example, those derived from the glucoamylase genefrom Schwanniomyces occidentalis, α-factor mating type gene fromSaccharomyces cerevisiae and the TAKA-amylase from Aspergillus oryzae.Alternatively, the PS4 nucleic acid may encode a fusion proteincomprising a membrane binding domain.

Expression Vector

The PS4 nucleic acid may be expressed at the desired levels in a hostorganism using an expression vector.

An expression vector comprising a PS4 nucleic acid can be any vectorwhich is capable of expressing the gene encoding PS4 nucleic acid in theselected host organism, and the choice of vector will depend on the hostcell into which it is to be introduced. Thus, the vector can be anautonomously replicating vector, i.e. a vector that exists as anepisomal entity, the replication of which is independent of chromosomalreplication, such as, for example, a plasmid, a bacteriophage or anepisomal element, a minichromosome or an artificial chromosome.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome.

Components of the Expression Vector

The expression vector typically includes the components of a cloningvector, such as, for example, an element that permits autonomousreplication of the vector in the selected host organism and one or morephenotypically detectable markers for selection purposes. The expressionvector normally comprises control nucleotide sequences encoding apromoter, operator, ribosome binding site, translation initiation signaland optionally, a repressor gene or one or more activator genes.Additionally, the expression vector may comprise a sequence coding foran amino acid sequence capable of targeting the PS4 variant polypeptideto a host cell organelle such as a peroxisome or to a particular hostcell compartment. Such a targeting sequence includes but is not limitedto the sequence SKL. In the present context, the term “expressionsignal” includes any of the above control sequences, repressor oractivator sequences. For expression under the direction of controlsequences, the nucleic acid sequence the PS4 variant polypeptide isoperably linked to the control sequences in proper manner with respectto expression.

Preferably, a polynucleotide in a vector is operably linked to a controlsequence that is capable of providing for the expression of the codingsequence by the host cell, i.e. the vector is an expression vector. Theterm “operably linked” means that the components described are in arelationship permitting them to function in their intended manner. Aregulatory sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved undercondition compatible with the control sequences.

The control sequences may be modified, for example by the addition offurther transcriptional regulatory elements to make the level oftranscription directed by the control sequences more responsive totranscriptional modulators. The control sequences may in particularcomprise promoters.

Promoter

In the vector, the nucleic acid sequence encoding for the variant PS4polypeptide is operably combined with a suitable promoter sequence. Thepromoter can be any DNA sequence having transcription activity in thehost organism of choice and can be derived from genes that arehomologous or heterologous to the host organism.

Bacterial Promoters

Examples of suitable promoters for directing the transcription of themodified nucleotide sequence, such as PS4 nucleic acids, in a bacterialhost include the promoter of the lac operon of E. coli, the Streptomycescoelicolor agarase gene dagA promoters, the promoters of the Bacilluslicheniformis α-amylase gene (amyL), the promoters of the Bacillusstearothermophilus maltogenic amylase gene (amyM), the promoters of theBacillus amyloliquefaciens α-amylase gene (amyQ), the promoters of theBacillus subtilis xylA and xylB genes and a promoter derived from aLactococcus sp.-derived promoter including the P170 promoter. When thegene encoding the PS4 variant polypeptide is expressed in a bacterialspecies such as E. coli, a suitable promoter can be selected, forexample, from a bacteriophage promoter including a T7 promoter and aphage lambda promoter.

Fungal Promoters

For transcription in a fungal species, examples of useful promoters arethose derived from the genes encoding the, Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral α-amylase, A. niger acid stable α-amylase, A. nigerglucoamylase, Rhizomucor miehei lipase, Aspergillus oryzae alkalineprotease, Aspergillus oryzae triose phosphate isomerase or Aspergillusnidulans acetamidase.

Yeast Promoters

Examples of suitable promoters for the expression in a yeast speciesinclude but are not limited to the Gal 1 and Gal 10 promoters ofSaccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters.

Host Organisms

(I) Bacterial Host Organisms

Examples of suitable bacterial host organisms are gram positivebacterial species such as Bacillaceae including Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus lautus, Bacillus megaterium and Bacillusthuringiensis, Streptomyces species such as Streptomyces murinus, lacticacid bacterial species including Lactococcus spp. such as Lactococcuslactis, Lactobacillus spp. including Lactobacillus reuteri, Leuconostocspp., Pediococcus spp. and Streptococcus spp. Alternatively, strains ofa gram-negative bacterial species belonging to Enterobacteriaceaeincluding E. coli or to Pseudomonadaceae can be selected as the hostorganism.

(II) Yeast Host Organisms

A suitable yeast host organism can be selected from thebiotechnologically relevant yeasts species such as but not limited toyeast species such as Pichia sp., Hansenula sp or Kluyveromyces,Yarrowinia species or a species of Saccharomyces including Saccharomycescerevisiae or a species belonging to Schizosaccharomyce such as, forexample, S. Pombe species.

Preferably a strain of the methylotrophic yeast species Pichia pastorisis used as the host organism. Preferably the host organism is aHansenula species.

(III) Fungal Host Organisms

Suitable host organisms among filamentous fungi include species ofAspergillus, e.g. Aspergillus niger, Aspergillus oryzae, Aspergillustubigensis, Aspergillus awamori or Aspergillus nidulans. Alternatively,strains of a Fusarium species, e.g. Fusarium oxysporum or of aRhizomucor species such as Rhizomucor miehei can be used as the hostorganism. Other suitable strains include Thermomyces and Mucor species.

Protein Expression and Purification

Host cells comprising polynucleotides may be used to expresspolypeptides, such as variant PS4 polypeptides, fragments, homologues,variants or derivatives thereof. Host cells may be cultured undersuitable conditions which allow expression of the proteins. Expressionof the polypeptides may be constitutive such that they are continuallyproduced, or inducible, requiring a stimulus to initiate expression. Inthe case of inducible expression, protein production can be initiatedwhen required by, for example, addition of an inducer substance to theculture medium, for example dexamethasone or IPTG.

Polypeptides can be extracted from host cells by a variety of techniquesknown in the art, including enzymatic, chemical and/or osmotic lysis andphysical disruption. Polypeptides may also be produced recombinantly inan in vitro cell-free system, such as the TnT™ (Promega) rabbitreticulocyte system.

EXAMPLES Example 1 Cloning of PS4

Pseudomonas saccharophila is grown overnight on LB media and chromosomalDNA is isolated by standard methods (Sambrook J, 1989). A 2190 bpfragment containing the PS4 open reading frame (Zhou et al., 1989) isamplified from P. sacharophila chromosomal DNA by PCR using the primersP1 and P2 (see Table 3). The resulting fragment is used as a template ina nested PCR with primers P3 and P4, amplifying the openreading frame ofPS4 without its signal sequence and introducing a NcoI site at the 5′end of the gene and a BamHI site at the 3′ end. Together with the NcoIsite a codon for a N-terminal Methionine is introduced, allowing forintracellular expression of PS4. The 1605 bp fragment is cloned intopCRBLUNT TOPO (Invitrogen) and the integrity of the construct analysedby sequencing. The E. coli Bacillus shuttle vector pDP66K (Penning a etal., 1996) is modified to allow for expression of the PS4 under controlof the P32 promoter and the ctgase signal sequence. The resultingplasmid, pCSmta is transformed into B. subtilis.

A second expression construct is made in which the starch binding domainof PS4 is removed. In a PCR with primers P3 and P6 (Table 3) on pCSmta,a truncated version of the mta gene is generated. The full length mtagene in pCSmta is exchanged with the truncated version which resulted inthe plasmid pCSmta-SBD.

Example 2 Site Directed Mutagenesis of PS4

Mutations are introduced into the mta gene by 2 methods. Either by a 2step PCR based method, or by a Quick Exchange method (QE). Forconvenience the mta gene is split up in 3 parts; a PvuI-FspI fragment, aFspI-PstI fragment and a PstI-AspI fragment, further on referred to asfragment 1, 2 and 3 respectively.

In the 2 step PCR based method, mutations are introduced using Pfu DNApolymerase (Stratagene). A first PCR is carried out with a mutagenesisprimer (Table 4) for the coding strand plus a primer downstream on thelower strand (either 2R or 3R Table 3). The reaction product is used asa primer in a second PCR together with a primer upstream on the codingstrand. The product of the last reaction is cloned into pCRBLUNT topo(Invitrogen) and after sequencing the fragment is exchanged with thecorresponding fragment in pCSmta.

Using the Quick Exchange method (Stratagene), mutations are introducedusing two complementary primers in a PCR on a plasmid containing the mtagene, or part of the mta gene.

For this purpose a convenient set of plasmids is constructed, comprisingof 3 SDM plasmids and 3 pCSΔ plasmids. The SDM plasmids each bear 1 ofthe fragments of the mta gene as mentioned above, in which the desiredmutation is introduced by QE. After verification by sequencing, thefragments are cloned into the corresponding recipient pCSΔ plasmid. ThepCSΔ plasmids are inactive derivatives from pCSmta. Activity is restoredby cloning the corresponding fragment from the SDM plasmid, enablingeasy screening.

TABLE 3 Primers used in cloning the mta gene, andstandard primers used in construction of sitedirected mutants with the 2 step PCR method. Intro- duced  PrimerPrimer sequence site P1 5′-ATG ACG AGG TCC TTG TTT TTC P25′-CGC TAG TCG TCC ATG TCG P3 5′-GCC ATG GAT CAG GCC GGC AAG AGC  NcoICCG P4 5′-TGG ATC CTC AGA ACG AGC CGC TGG T BamHI P65′-GAA TTC AGC CGC CGT CAT TCC CGC C EcoRI 2L5′-AGA TTT ACG GCA TGT TTC GC 2R 5′-TAG CCG CTA TGG AAG CTG AT 3L5′-TGA CCT TCG TCG ACA ACC AC 3R 5′-GAT AGC TGC TGG TGA CGG TC

TABLE 4 Primers used to introduce site directed mutations in mta Modifi-Pur- Mutation Oligo Sequence cation Strand pose G134RCTGCCGGCCGGCCAGcGCTTCT + SDM GGCG G134R − cgccagaagcgctggccggccg − SDMgcag I157L GACGGTGACCGCTTCcTgGGCG + SDM GCGAGTCG I151L −cgactcgccgcccaggaagcgg − SDM tcaccgtc G223A GGCGAGCTGTGGAAAgccCCTT + SDMCTGAATATCCG G223A − cggatattcagaaggggctttc − SDM cacagctcgcc H307LgaacGGCGGCCAGCACctgTGG + SDM GCGCTGCAG H307L − ctgcagcgcccacaggtgctgg −SDM ccgccgttc S334P, GTACTGGccgCACATGTACGAC + SDM D343ETGGGGCTACGGCgaaTTCATC S334P, gatgaattcgccgtagccccag − SDM D343E −tcgtacatgtgcggccagtac

TABLE 5 Features of the SDM and pCSΔ plasmids PlasmidFeatures/construction SDM1 pBlueSK + 480 bp SalI-StuI fragment mta SDM2pBlueSK + 572 bp SacII-PstI fragment mta SDM3 pBlueSK +471 bp SalI-StuI fragment mta pCSΔ1FseI site filled in with Klenow ----> frameshift in mta pCSΔ2FspI-PstI fragment of mta replaced with ‘junk-DNA’ pCSΔ3PstI-AspI fragment of mta replaced with ‘junk-DNA’

Example 3 Multi SDM

The PS4 variants were generated using a QuickChange® Multi Site DirectedMutagenesis Kit (Stratagene) according to the manufactures protocol withsome modifications as described.

Step 1: Mutant Strand Synthesis Reaction (PCR)

Inoculate 3 ml. LB (22 g/l Lennox L Broth Base, Sigma)+antibiotics (0.05μg/ml kanamycin, Sigma) in a 10 ml Falcon tube

-   -   Incubate o/n 37° C., ca. 200 rpm.    -   Spin down the cells by centrifugation (5000 rpm/5 min)    -   Poor off the medium    -   Prepare ds-DNA template using QIAGEN Plasmid Mini Purification        Protocol

1. The mutant strand synthesis reaction for thermal cycling was preparedas follow:

PCR Mix:

2.5 μl 10× QuickChange ® Multi reaction buffer 0.75 μl QuickSolution Xμl

1 μl dNTP mix X μl ds-DNA template (200 ng) 1 μl QuickChange ® Multienzyme blend (2.5 U/μl) (PfuTurbo ® DNA polymerase) X μl dH₂O (to afinal volume of 25 μl)

Mix all components by pipetting and briefly spin down the reactionmixtures.

2. Cycle the reactions using the following parameters:

-   -   35 cycles of denaturation (96° C./1 min)        -   primer annealing (62.8° C./1 min)        -   elongation (65° C./15 min)        -   then hold at 4° C.    -   Preheat the lid of the PCR machine to 105° C. and the plate to        95° C. before the PCR tubes are placed in the machine (eppendorf        thermal cycler).        Step 2: Dpn I Digestion    -   1. Add 2 pd Dpn I restriction enzyme (10 U/μl) to each        amplification reaction, mix by pipetting and spin down mixture.    -   2. Incubate at 37° C. for ˜3 hr.        Step 3: Transformation of XL10-Gold® Ultracompetent Cells    -   1. Thaw XL10Gold cells on ice. Aliquot 45 μl cells per        mutagenesis reaction to prechilled Falcon tubes.    -   2. Turn on the waterbath (42° C.) and place a tube with NZY⁺        broth in the bath to preheat.    -   3. Add 2 μl β-mercaptoethanol mix to each tube. Swirl and tap        gently and incubate 10 min on ice, swirling every 2 min.    -   4. Add 1.5 μl Dpn I-treated DNA to each aliquot of cells, swirl        to mix and incubate on ice for 30 min.    -   5. Heat-pulse the tubes in 42° C. waterbath for 30 s and place        on ice for 2 min.    -   6. Add 0.5 ml preheated NZY⁺ broth to each tube and incubate at        37° C. for 1 hr with shaking at 225-250 rpm.    -   7. Plate 200 μl of each transformation reaction on LB plates        (33.6 g/l Lennox L Agar, Sigma) containing 1% starch and 0.05        μg/ml kanamycin    -   8. Incubate the transformation plates at 37° C. overnight.

TABLE 6 Primer table for pPD77d14: Muta- Modifi- Pur- tionOligo Sequence cation Strand pose N33Y, GCGAAGCGCCCTACAACTGGTA 5′phos- + MSDM D34N CAAC phate K71R CCGACGGCGGCAGGTCCGGCG 5′ phos- + MSDMphate G87S CAAGAACAGCCGCTACGGCAGC 5′ phos- + MSDM GAC phate G121DCACATGAACCGCGACTACCCGG 5′ phos- + MSDM ACAAG phate G134RCTGCCGGCCGGCCAGcGCTTCT 5′ phos- + MSDM GGCG phate A141PCGCAACGACTGCGCCGACCCGG 5′ phos- + MSDM G phate I157LGACGGTGACCGCTTCcTgGGCG 5′ phos- + MSDM GCGAGTCG phate L178F,CGCGACGAGTTTACCAACCTGC 5′ phos- + MSDM A179T G phate G223AGGCGAGCTGTGGAAAgccCCTT 5′ phos- + MSDM CTGAATATCCG phate H307LgaacGGCGGCCAGCACctgTGG 5′ phos- + MSDM GCGCTGCAG phate S334P,GTACTGGccgCACATGTACGAC 5′ phos- + MSDM D343E TGGGGCTACGGCgaaTTCATC phate

TABLE 7 Primer table for pPD77d20: Muta- Modifi- Pur- tionOligo Sequence cation Strand pose N33Y, GCGAAGCGCCCTACAACTGGTA 5′phos- + MSDM D34N CAAC phate K71R CCGACGGCGGCAGGTCCGGCG 5′ phos- + MSDMphate G121D CACATGAACCGCGACTACCCGG 5′ phos- + MSDM ACAAG phate G134RCTGCCGGCCGGCCAGcGCTTCT 5′ phos- + MSDM GGCG phate A141PCGCAACGACTGCGCCGACCCGG 5′ phos- + MSDM G phate I157LGACGGTGACCGCTTCcTgGGCG 5′ phos- + MSDM GCGAGTCG phate L178F,CGCGACGAGTTTACCAACCTGC 5′ phos- + MSDM A179T G phate G223AGGCGAGCTGTGGAAAgccCCTT 5′ phos- + MSDM CTGAATATCCG phate H307LgaacGGCGGCCAGCACctgTGG 5′ phos- + MSDM GCGCTGCAG phate S334P,GTACTGGccgCACATGTACGAC 5′ phos- + MSDM D343E TGGGGCTACGGCgaaTTCATC phate

TABLE 8 Primer table for pPD77d34: Muta- Modifi- Pur- tionOligo Sequence cation Strand pose N33Y, GCGAAGCGCCCTACAACTGGTA 5′phos- + MSDM D34N CAAC phate G121D CACATGAACCGCGACTACCCGG 5′ phos- +MSDM ACAAG phate G134R CTGCCGGCCGGCCAGcGCTTCT 5′ phos- + MSDM GGCG phateA141P CGCAACGACTGCGCCGACCCGG 5′ phos- + MSDM G phate I157LGACGGTGACCGCTTCcTgGGCG 5′ phos- + MSDM GCGAGTCG phate L178F,CGCGACGAGTTTACCAACCTGC 5′ phos- + MSDM A179T G phate G223AGGCGAGCTGTGGAAAgccCCTT 5′ phos- + MSDM CTGAATATCCG phate H307LgaacGGCGGCCAGCACctgTGG 5′ phos- + MSDM GCGCTGCAG phate S334PGTACTGGccgCACATGTACGAC 5′ phos- + MSDM TGGGGCTACGGC phateVector System based on pPD77The vector system used for pPD77 is based on pCRbluntTOPOII(invitrogen). The zeocin resistance cassette has been removed by pmII,393 bp fragment removed. The expression cassette from the pCC vector(P32-ssCGTase-PS4-tt) has then been inserted into the vector.Ligation of PS4 Variant into pCCMiniThe plasmid which contain the relevant mutations (created by MSDM) iscut with restriction enzyme Nco 1 and Hind III (Biolabs):

-   -   3 μg plasmid DNA, X μl 10× buffer 2, 10 units NcoI, 20 units        HindIII, Incubation 2 h at 37° C.        Run digestion on a 1% agarose gel. Fragments sized 1293 bp (PS4        gene) is cut out of the gel and purified using Qiagen gel        purification kit.        The vector pCCMini is then cut with restriction enzymes, Nco 1        and Hind III, and the digestion is then run on a 1% agarose gel.        The fragment sized 3569 bp is cut out of the gel and purified        using Qiagen gel purification kit.        Ligation: Use Rapid DNA ligation kit (Roche)        Use the double amount of insert compared to vector    -   e.g. 2 μl insert (PS4 gene)        -   1 μl vector        -   5 μl T4 DNA ligation buffer 2× conc        -   1 μl dH₂O        -   1 μl T4 DNA ligase            Ligate 5 min/RT            Transform the ligation into One Shot TOPO competent cells            according to manufactures protocol (Invitrogen). Use 5 μl            ligation pr. transformation.            Plate 50 μl transformations mix onto LB plates (33.6 g/l            Lennox L Agar, Sigma) containing 1% starch and 0.05 μg/ml            kanamycin. Vectors containing insert (PS4 variants) can be            recognised by halo formation on the starch plates.

Example 4 Transformation into Bacillus subtilis (ProtoplastTransformation)

Bacillus subtilis (strain DB104A; Smith et al. 1988; Gene 70, 351-361)is transformed with the mutated pCS-plasmids according to the followingprotocol.

A. Media for Protoplasting and Transformation

2 x SMM per litre: 342 g sucrose (1 M); 4.72 g sodium maleate (0.04 M);8:12 g MgCl₂, 6H₂0 (0.04 M); pH 6.5 with concentrated NaOH. Distributein 50-ml portions and autoclave for 10 min. 4 x YT 2 g Yeast extract +3.2 g Tryptone + 0.5 g NaCl per (1/2 NaCl) 100 ml. mix equal volumes of2 × SMM and 4 33 YT. SMMP 10 g polyethyleneglycol 6000 (BDH) or 8000(Sigma) in PEG 25 ml 1 x SMM (autoclave for 10 min.).

B. Media for Plating/Regeneration

agar 4% Difco minimal agar. Autoclave for 15 min. sodium succinate 270g/l (1 M), pH 7.3 with HCl. Autoclave for 15 min. phosphate buffer 3.5 gK₂HPO₄ + 1.5 g KH₂PO₄ per 100 ml. Autoclave for 15 min. MgCl₂ 20.3 gMgCl₂, 6H₂O per 100 ml (1 M). casamino acids 5% (w/v) solution.Autoclave for 15 min. yeast extract 10 g per 100 ml, autoclave for 15min. glucose 20% (w/v) solution. Autoclave for 10 min.

DM3 regeneration medium: mix at 60 C (waterbath; 500-ml bottle):

-   -   250 ml sodium succinate    -   50 ml casamino acids    -   25 ml yeast extract    -   50 ml phosphate buffer    -   15 ml glucose    -   10 ml MgCl₂    -   100 ml molten agar        Add appropriate antibiotics: chloramphenicol and tetracycline, 5        ug/ml; erythromycin, 1 ug/ml. Selection on kanamycin is        problematic in DM3 medium: concentrations of 250 ug/ml may be        required.

C. Preparation of Protoplasts

1. Use detergent-free plastic or glassware throughout.

2. Inoculate 10 ml of 2×YT medium in a 100-ml flask from a singlecolony. Grow an overnight culture at 25-30 C in a shaker (200 rev/min).

3. Dilute the overnight culture 20 fold into 100 ml of fresh 2×YT medium(250-ml flask) and grow until OD₆₀₀=0.4−0.5 (approx. 2 h) at 37 C in ashaker (200-250 rev/min).

4. Harvest the cells by centrifugation (9000 g, 20 min, 4 C).

5. Remove the supernatant with pipette and resuspend the cells in 5 mlof SMMP+5 mg lysozyme, sterile filtered.

6. Incubate at 37 C in a waterbath shaker (100 rev/min).

After 30 min and thereafter at 15 min intervals, examine 25 ul samplesby microscopy. Continue incubation until 99% of the cells areprotoplasted (globular appearance). Harvest the protoplasts bycentrifugation (4000 g, 20 min, RT) and pipet off the supernatant.Resuspend the pellet gently in 1-2 ml of SMMP.

The protoplasts are now ready for use. (Portions (e.g. 0.15 ml) can befrozen at −80 C for future use (glycerol addition is not required).Although this may result in some reduction of transformability, 106transformants per ug of DNA can be obtained with frozen protoplasts).

D. Transformation

1. Transfer 450 ul of PEG to a microtube.

2. Mix 1-10 ul of DNA (0.2 ug) with 150 ul of protoplasts and add themixture to the microtube with PEG. Mix immediately, but gently.

3. Leave for 2 min at RT, and then add 1.5 ml of SMMP and mix.

4. Harvest protoplasts by microfuging (10 min, 13.000 rev/min (10-12.000g)) and pour off the supernatant. Remove the remaining droplets with atissue.

Add 300 ul of SMMP (do not vortex) and incubate for 60-90 min at 37 C ina waterbath shaker (100 rev/min) to allow for expression of antibioticresistance markers. (The protoplasts become sufficiently resuspendedthrough the shaking action of the waterbath.). Make appropriatedilutions in 1×SSM and plate 0.1 ml on DM3 plates

Example 5 Fermentation of PS4 Variants in Shake Flasks

The shake flask substrate is prepared as follows:

Ingredient %(w/v) Water — Yeast extract 2 Soy Flour 2 NaCl 0.5Dipotassium phosphate 0.5 Antifoam agent 0.05

The substrate is adjusted to pH 6.8 with 4N sulfuric acid or sodiumhydroxide before autoclaving. 100 ml of substrate is placed in a 500 mlflask with one baffle and autoclaved for 30 minutes. Subsequently, 6 mlof sterile dextrose syrup is added. The dextrose syrup is prepared bymixing one volume of 50% w/v dextrose with one volume of water followedby autoclaving for 20 minutes.

The shake flasks are inoculated with the variants and incubated for 24hours at 35° C./180 rpm in an incubator. After incubation cells areseparated from broth by centrifugation (10.000×g in 10 minutes) andfinally, the supernatant is made cell free by microfiltration at 0.2 cm.The cell free supernatant is used for assays and application tests.

Example 6 Amylase Assays

Betamyl Assay

One Betamyl unit is defined as activity degrading 0.0351 mmole per 1 minof PNP-coupled maltopentaose so that 0.0351 mmole PNP per 1 min. can bereleased by excess a-glucosidase in the assay mix. The assay mixcontains 50 ul 50 mM Na-citrate, 5 mM CaCl2, pH 6.5 with 25 ul enzymesample and 25 ul Betamyl substrate (Glc5-PNP and a-glucosidase) fromMegazyme, Ireland (1 vial dissolved in 10 ml water). The assay mix isincubated for 30 min. at 40 C and then stopped by adding 150 ul 4% Tris.Absorbance at 420 nm is measured using an ELISA-reader and the Betamylactivity is calculate based on Activity=A420*d in Betamyl units/ml ofenzyme sample assayed.

Endo-Amylase Assay

The endo-amylase assay is identical to the Phadebas assay run accordingto manufacturer (Pharmacia & Upjohn Diagnostics AB).

Exo-Specificity

The ratio of exo-amylase activity to Phadebas activity was used toevaluate exo-specificity.

Specific Activity

For the PSac-D14, PSac-D20 and PSac-D34 variants we find an averagespecific activity of 10 Betamyl units per microgram of purified proteinmeasured according to Bradford (1976; Anal. Biochem. 72, 248). Thisspecific activity is used for based on activity to calculate the dosagesused in the application trials.

Example 7 Half-Life Determination

t1/29 is defined as the time (in minutes) during which half the enzymeactivity is inactivated under defined heat conditions. In order todetermine the half life of the enzyme, the sample is heated for 1-10minutes at constant temperatures of 60° C. to 90° C. The half life iscalculated based on the residual Betamyl assay.

Procedure: In an Eppendorf vial, 1000 μl buffer is preheated for atleast 10 minutes at 60° C. or higher. The heat treatment of the sampleis started addition of 100 μL of the sample to the preheated bufferunder continuous mixing (800 rpm) of the Eppendorf vial in an heatincubator (Termomixer comfort from Eppendorf). After 0, 2, 4, 6, 8 and 9minutes of incubation, the treatment is stopped by transferring 45 μl ofthe sample to 1000 μl of the buffer equilibrated at 20° C. andincubating for one minute at 1500 rpm and at 20° C. The residualactivity is measured with the Betamyl assay.

Calculation: Calculation of t1/2 is based on the slope of log 10 (thebase-10 logarithm) of the residual Betamyl activity versus theincubation time. t1/2 is calculated as Slope/0.301=t1/2.

Example 8 Model System Baking Tests

The doughs are made in the Farinograph at 30.0° C. 10.00 g reformedflour is weighed out and added in the Farinograph; after 1 min. mixingthe reference/sample (reference=buffer or water, sample=enzyme+buffer orwater) is added with a sterile pipette through the holes of the kneadingvat After 30 sec. the flour is scraped off the edges—also through theholes of the kneading vat. The sample is kneaded for 7 min.

A test with buffer or water is performed on the Farinograph before thefinal reference is run. FU should be 400 on the reference, if it is not,this should be adjusted with, for example, the quantity of liquid. Thereference/sample is removed with a spatula and placed in the hand (witha disposable glove on it), before it is filled into small glass tubes(of approx. 4.5 cm's length) that are put in NMR tubes and corked up. 7tubes per dough are made.

When all the samples have been prepared, the tubes are placed in a(programmable) water bath at 33° C. (without corks) for 25 min. andhereafter the water bath is set to stay for 5 min. at 33° C., then toheated to 98° C. over 56 min. (1.1° C. per minute) and finally to stayfor 5 min. at 96° C.

The tubes are stored at 20.0° C. in a thermo cupboard. The solid contentof the crumb was measured by proton NMR using a Bruker NMS 120 MinispecNMR analyser at day 1, 3 and 7 as shown for crumb samples prepared with0, 05, 1 and 2 ppm PSacD34 in FIG. 2. The lower increase in solidcontent over time represents the reduction in amylopectinretrogradation. After 7 days of storage at 20.0° C. in a thermo cupboard10-20 mg samples of crumb weighed out and placed in 40 μl aluminiumstandard DSC capsules and kept at 20° C.

The capsules are used for Differential Scanning Calorimetry on a MettlerToledo DSC 820 instrument. As parameters are used a heating cycle of20-95° C. with 10° C. per min. heating and Gas/flow: N₂/80 ml per min.The results are analysed and the enthalpy for melting of retrogradedamylopectin is calculated in J/g.

Example 9 Antistaling Effects

Model bread crumbs are prepared and measured according to Example 8. Asshown in Table 2, PS4 variants show a strong reduction of theamylopectin retrogradation after baking as measured by DifferentialScanning Calorimetry in comparison to the control. The PS4 variants showa clear dosage effect.

Example 10 Firmness Effects in Baking Trials

Baking trials were carried out with a standard white bread sponge anddough recipe for US toast. The sponge dough is prepared from 1600 g offlour “All Purpose Classic” from Sisco Mills, USA”, 950 g of water, 40 gof soy bean oil and 32 g of dry yeast. The sponge is mixed for 1 min. atlow speed and subsequently 3 min. at speed 2 on a Hobart spiral mixer.The sponge is subsequently fermented for 2.5 hours at 35° C., 85% RHfollowed by 0.5 hour at 5° C.

Thereafter 400 g of flour, 4 g of dry yeast, 40 g of salt, 2.4 g ofcalcium propionate, 240 g of high fructose corn sirup (Isosweet), 5 g ofthe emulsifier PANODAN 205, 5 g of enzyme active soy flour, 30 g ofnon-active soy flour, 220 g of water and 30 g of a solution of ascorbicacid (prepared from 4 g ascorbic acid solubilised in 500 g of water) areadded to the sponge. The resulting dough is mixed for 1 min. at lowspeed and then 6 min. on speed 2 on a Diosna mixer. Thereafter the doughis rested for 5 min. at ambient temperature, and then 550 g dough piecesare scaled, rested for 5 min. and then sheeted on Glimek sheeter withthe settings 1:4, 2:4, 3:15, 4:12 and 10 on each side and transferred toa baking form. After 60 min. proofing at 43° C. at 90% RH the doughs arebaked for 29 min. at 218° C.

Firmness and resilience were measured with a TA-XT 2 texture analyser.The Softness, cohesiveness and resilience is determined by analysingbread slices by Texture Profile Analysis using a Texture Analyser FromStable Micro Systems, UK. The following settings were used:

Pre Test Speed: 2 mm/s

Test Speed: 2 mm/s

Post Test Speed: 10 mm/s

Rupture Test Distance: 1%

Distance: 40%

Force: 0.098 N

Time: 5.00 sec

Count: 5

Load Cell: 5 kg

Trigger Type Auto—0.01 N

Firmness measurements show that the PS4 variant polypeptidessignificantly reduce the firmness development from day 1 to day 7 andshow a higher effect with increasing enzyme dosage.

Example 11 Control of Volume of Danish Rolls

Danish Rolls are prepared from a dough based on 2000 g Danish reformflour (from Cerealia), 120 g compressed yeast, 32 g salt, and 32 gsucrose. Water is added to the dough according to prior wateroptimisation.

The dough is mixed on a Diosna mixer (2 min. at low speed and 5 min athigh speed). The dough temperature after mixing is kept at 26° C. 1350 gdough is scaled and rested for 10 min. in a heating cabinet at 30° C.The rolls are moulded on a Fortuna molder and proofed for 45 min. at 34°C. and at 85% relative humidity. Subsequently the rolls are baked in aBago 2 oven for 18 min. at 250° C. with steam in the first 13 seconds.After baking the rolls are cooled for 25 min. before weighing andmeasuring of volume.

The rolls are evaluated regarding crust appearance, crumb homogeneity,capping of the crust ausbund and specific volume (measuring the volumewith the rape seed displacement method).

Based on these criteria it is found that the PS4 variants increase thespecific volume and improve the quality parameters of Danish rolls. ThusPS4 variants are able to control the volume of baked products.

Example 12 Results

TABLE 9 Biochemical properties of PSac-variants compared to wild-typePSac-cc1 t½- Betamyl/ Variant t½-75 80 Phadebas Mutations PSac-cc1 <0.540 PSac-D3 9.3 3 43 N33Y, D34N, K71R, G134R, A141P, I157L, L178F, A179T,G223A, H307L, D343E, S334P PSac-D14 9.3 2.7 65 N33Y, D34N, K71R, G87S,G121D, (SEQ ID G134R, A141P, I157L, L178F, NO: 4) A179T, G223A, H307L,D343E, S334P PSac-D20 7.1 2.7 86 N33Y, D34N, K71R, G121D, (SEQ ID G134R,A141P, I157L, L178F, NO: 3) A179T, G223A, H307L, D343E, S334P PSac-D348.4 2.9 67 N33Y, D34N, G121D, G134R, (SEQ ID A141P, I157L, L178F, A179T,NO: 2) G223A, H307L, S334P PSac- 7.1 3 51 N33Y, D34N, G134R, A141P,pPD77d33 I157L, L178F, A179T, G223A, (SEQ ID H307L, S334P NO: 13) pMD556.0 54 N33Y D34N G121F G134R, A141P I157L G223A H307L S334P L178F A179TpMD85 5.1 115 N33Y D34N G121F G134R, A141P I157L G223E H307L S334P L178FA179T PMD96 4.0 231 N33Y D34N G121F G134R, A141P I157L G223E H307L S334PL178F A179T S161A pMD86 3.6 170 N33Y D34N G121A G134R, A141P I157L G223EH307L S334P L178F A179T pMD109 3.6 170 N33Y D34N G121A G134R, A141PI157L G223E H307L S334P L178F A179T S161A

Sequences pPD77d40, pMD55, pMD85, pMD96, pMD86 and pMD109 have theresidues at column 5 mutated and the starch binding domain deleted in aP. saccharophila wild type background (SEQ ID NO: 1). Their sequencesmay be constructed in a straightforward manner with this information.

The detailed effects of individual mutations at various positions aredescribed in the following subsections.

Enhanced Thermostability of PS4 Variant Polypeptide with Mutation 121F

A PS4 variant polypeptide designated pMD55 having amino acid mutationsat N33Y, D34N, G121F, G134R, A141P, I157L, G223A, H307L, S334P, L178F,A179T is tested for thermostability. This polypeptide displays improvedthermostability as shown in the table below. See also Example 12 andTable 9 above.

Betamyl/ Variant t½-75 t½-80 Phadebas Mutations PSac- 7.1 3 51 N33Y D34NG134R A141P I157L pPD77d33 L178F A179T G223A H307L (SEQ ID S334P NO: 13)pMD55 6.0 54 N33Y D34N G121F G134R A141P I157L L178F A179T G223A H307LS334P

Enhanced Exo-Specificity of PS4 Variant Polypeptide with Mutation 161A

A PS4 variant polypeptide designated pMD96 having amino acid mutationsat N33Y, D34N, G121F, G134R, A141P, I157L, S161A, L178F, A179T, G223E,H307L, S334P is tested for exo-specificity. This polypeptide displaysimproved exo-specificity as shown in the table below. See also Example12 and Table 9 above.

Betamyl/ Variant t½-75 t½-80 Phadebas Mutations pMD85 5.1 115 N33Y D34NG121F G134R A141P I157L L178F A179T G223E H307L S334P PMD96 4.0 231 N33YD34N G121F G134R A141P I157L S161A L178F A179T G223E H307L S334P

Enhanced Exo-Specificity of PS4 Variant Polypeptide with Mutation 223E

A PS4 variant polypeptide designated pMD85 having amino acid mutationsat N33Y, D34N, G121F, 0134R, A141P, I157L, L178F, A179T, G223E, H307L,S334P is tested for exo-specificity. This polypeptide displays improvedexo-specificity as shown in the table below. See also Example 12 andTable 9 above.

Betamyl/ Variant t½-75 t½-80 Phadebas Mutations pMD55 6.0 54 N33Y D34NG121F G134R A141P I157L L178F A179T G223A H307L S334P pMD85 5.1 115 N33YD34N G121F G134R, A141P I157L L178F A179T G223E H307L S334P

Example 13 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 121D

A PS4 variant polypeptide designated pMD3 having amino acid mutations atN33Y D34N G134R A141P I157L L178F A179T G121D H307L S334P is tested forthermostability and exo-specificity. This polypeptide displays improvedthermostability and improved exospecificity as shown in the table below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD28 2.3 41 N33Y D34N G134RA141P I157L L178F A179T G223A H307L S334P pMD3 2.8 99 N33Y D34N G134RA141P I157L L178F A179T G121D H307L S334P

Example 14 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 121W

A PS4 variant polypeptide designated pMD44 having amino acid mutationsat N33Y D34N G134R A141P I157L L178F A179T G121W H307L S334P is testedfor thermostability and exo-specificity. This polypeptide displaysimproved thermostability and improved exo-specificity as shown in thetable below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD28 2.3 41 N33Y D34N G134RA141P I157L L178F A179T G223A H307L S334P pMD44 4.8 172 N33Y D34N G134RA141P I157L L178F A179T G121W H307L S334P

Example 15 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 121H

A PS4 variant polypeptide designated pMD43 a having amino acid mutationsat N33Y D34N G134R A141P I157L L178F A179T G121H H307L S334P is testedfor thermostability and exo-specificity. This polypeptide displaysimproved thermostability and improved exo-specificity as shown in thetable below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD28 2.3 41 N33Y D34N G134RA141P I157L L178F A179T G223A H307L S334P pMD43 a 3.7 110 N33Y D34NG134R A141P I157L L178F A179T G121H H307L S334P

Example 16 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 121M

A PS4 variant polypeptide designated pMD41 a having amino acid mutationsat N33Y D34N G134R A141P I157L L178F A179T G121M H307L S334P is testedfor thermostability and exo-specificity. This polypeptide displaysimproved thermostability and improved exo-specificity as shown in thetable below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD28 2.3 41 N33Y D34N G134RA141P I157L L178F A179T G223A H307L S334P pMD41 a 3.3 145 N33Y D34NG134R A141P I157L L178F A179T G121M H307L S334P

Example 17 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 121A

A PS4 variant polypeptide designated pMD74 a having amino acid mutationsat N33Y D34N G134R A141P I157L L178F A179T G121A H307L S334P is testedfor thermostability and exo-specificity. This polypeptide displaysimproved thermostability and improved exo-specificity as shown in thetable below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD28 2.3 41 N33Y D34N G134RA141P I157L L178F A179T G223A H307L S334P pMD74 a 3.3 87 N33Y D34N G134RA141P I157L L178F A179T G121A H307L S334P

Example 18 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 121Y

A PS4 variant polypeptide designated pMD73a having amino acid mutationsat N33Y D34N G134R A141P I157L L178F A179T G121Y H307L S334P is testedfor thermostability and exo-specificity. This polypeptide displaysimproved thermostability and improved exo-specificity as shown in thetable below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD28 2.3 41 N33Y D34N G134RA141P I157L L178F A179T G223A H307L S334P pMD73 4.8 101 N33Y D34N G134RA141P I157L L178F A179T G121Y H307L S334P

Example 19 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 223A

A PS4 variant polypeptide designated pMD3 having amino acid mutations atN33Y D34N G121D G134R A141P I157L L178F A179T G223A H307L S334P istested for thermostability and exo-specificity. This polypeptidedisplays improved thermostability and improved exo-specificity as shownin the table below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD25 1.3 58 N33Y D34N G121DG134R A141P I157L L178F A179T H307L S334P pMD3 2.8 99 N33Y D34N G121DG134R A141P I157L L178F A179T G223A H307L S334P

Example 20 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 223K

A PS4 variant polypeptide designated SSM173 F6 having amino acidmutations at N33Y D34N G121D G134R A141P I157L L178F A179T G223K H307LS334P is tested for thermostability and exo-specificity. Thispolypeptide displays improved thermostability and improvedexo-specificity as shown in the table below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD25 1.3 58 N33Y D34N G121DG134R A141P I157L L178F A179T H307L S334P SSM173 F6 1.6 118 N33Y D34NG121D G134R A141P I157L L178F A179T G223K H307L S334P

Example 21 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 223V

A PS4 variant polypeptide designated pMD49 a having amino acid mutationsat N33Y D34N G121D G134R A141P I157L L178F A179T G223V H307L S334P istested for thermostability and exo-specificity. This polypeptidedisplays improved thermostability and improved exo-specificity as shownin the table below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD25 1.3 58 N33Y D34N G121DG134R A141P I157L L178F A179T H307L S334P pMD49 a 0.6 115 N33Y D34NG121D G134R A141P I157L L178F A179T G223V H307L S334P

Example 22 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 223E

A PS4 variant polypeptide designated SSM171 G11 having amino acidmutations at N33Y D34N G121D G134R A141P I157L L178F A179T G223E H307LS334P is tested for thermostability and exo-specificity. Thispolypeptide displays improved thermostability and improvedexo-specificity as shown in the table below.

t½- Betamyl/ Variant 80 Phadebas Mutations pMD25 1.3 58 N33Y D34N G121DG134R A141P I157L L178F A179T H307L S334P SSM171 G11 3.0 113 N33Y D34NG121D G134R A141P I157L L178F A179T G223E H307L S334P

Example 23 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation G223R

A PS4 variant polypeptide designated SSM173 B6 having amino acidmutations at N33Y D34N G121D G134R A141P I157L L178F A179T G223R H307LS334P is tested for thermostability and exo-specificity. Thispolypeptide displays improved thermostability and improvedexo-specificity as shown in the table below.

Betamyl/ Variant t½-80 Phadebas Mutations pMD25 1.3 58 N33Y D34N G121DG134R A141P I157L L178F A179T H307L S334P SSM173 B6 1.5 76 N33Y D34NG121D G134R A141P I157L L178F A179T G223R H307L S334P

Example 24 Enhanced Thermostability of PS4 Variant Polypeptide withMutation Y146G

A PS4 variant polypeptide designated SSM 381 having amino acid mutationsat 33Y, 34N, 121F, 134R, 141P, 146G, 157L, 161A, 178F, 179T, 223E, 307Land 334P is tested for thermostability. This polypeptide displaysimproved thermostability as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone t½-80 t½-85 15 SSM381 Y146GpMD96 20 2.6 14 PMD96 6.0 1.4

Example 25 Enhanced Thermostability of PS4 Variant Polypeptide withMutation 157M

A PS4 variant polypeptide designated SSM279 B1 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157M, 161A, 179F, 179T, 223E,307L and 334P is tested for thermostability. This polypeptide displaysimproved thermostability as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone t½-80 16 PMD140 L157M PMD96 7.613 PMD96 6.0

Example 26 Enhanced Thermostability of PS4 Variant Polypeptide withMutation 158T

A PS4 variant polypeptide designated SSM237 P2 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 158T, 161A, 178F, 179T,223E, 307L and 334P is tested for thermostability. This polypeptidedisplays improved thermostability as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone t½-80 17 PMD130 G158T PMD96 8.013 PMD96 6.0

Example 27 Enhanced Thermostability of PS4 Variant Polypeptide withMutation 198W and/or 229P

A PS4 variant polypeptide designated SSM325 F3 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,229P, 307L and 334P is tested for thermostability. This polypeptidedisplays improved thermostability as shown in the table below.

A PS4 variant polypeptide designated pMD129 having amino acid mutationsat 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 198W, 223E, 229P,307L and 334P is tested for thermostability. This polypeptide displaysimproved thermostability as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone t½-80 t½-85 20 pMD129 Y198W,S229P pMD96 2.4 19 SSM325 F3 S229P pMD96 7.9 1.8 14 pMD96 6.0 1.4

Example 28 Enhanced Exo-Specificity of PS4 Variant Polypeptide withMutation G303E or G303D

A PS4 variant polypeptide designated SSM341 A9 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,303B, 307L and 334P is tested for exo-specificity. This polypeptidedisplays improved exo-specificity as shown in the table below.

A PS4 variant polypeptide designated SSM341 G11 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,303D, 307L and 334P is tested for exo-specificity. This polypeptidedisplays improved exo-specificity as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone Betamyl/Phadebas 21 SSM341 A9G303E pMD96 256 22 SSM341 G11 G303D pMD96 230 14 pMD96 179

Example 29 Enhanced Exo-Specificity of PS4 Variant Polypeptide withMutation H306T or H1306G

A PS4 variant polypeptide designated SSM350 B11 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,306T, 307L and 334P is tested for exo-specificity. This polypeptidedisplays improved exo-specificity as shown in the table below.

A PS4 variant polypeptide designated SSM350 C12 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,306G, 307L and 334P is tested for exo-specificity. This polypeptidedisplays improved exo-specificity as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone Betamyl/Phadebas 23 SSM350 B11H306T pMD96 271 24 SSM350 C12 H306G pMD96 195 14 pMD96 179

Example 30 Enhanced Exo-Specificity of PS4 Variant Polypeptide withMutation A309P

A PS4 variant polypeptide designated SSM332 Q4 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,309P, 307L and 334P is tested for thermostability. This polypeptidedisplays improved thermostability as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone t½-80 t½-85 25 SSM332 Q4 A309PpMD96 7.5 2.5 14 PMD96 6.0 1.4

Example 31 Enhanced Thermostability of PS4 Variant Polypeptide withMutation R316S or R316P

A PS4 variant polypeptide designated SSM365 B4 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,307L, 316S, and 334P is tested for thermostability. This polypeptidedisplays improved thermostability as shown in the table below.

A PS4 variant polypeptide designated SSM365 F4 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,307L, 316P and 334P is tested for thermostability. This polypeptidedisplays improved thermostability as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone t½-80 t½-85 26 SSM365 B4 R316SpMD96 7.5 2.5 27 SSM365 F4 R316P pMD96 7.1 2.0 14 PMD96 6.0 1.4

Example 32 Enhanced Thermostability of PS4 Variant Polypeptide withMutation R353T

A PS4 variant polypeptide designated SSM360 C7 having amino acidmutations at 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,307L, 334P and 353T is tested for thermostability. This polypeptidedisplays improved thermostability as shown in the table below.

SEQ ID NO: Identifier Mutation Backbone t½-80 t½-85 28 SSM360 C7 R353TpMD96 5.6 2.6 14 PMD96 6.0 1.4

Example 33 Enhanced Exo-Specificity of PS4 Variant Polypeptide withMutation 26E

A PS4 variant polypeptide designated SSM219 B3 having amino acidmutations at N26E N33Y D34N G121F G134R A141P I157L L178F A179T G223AH307L S334P is tested for exo-specificity. This polypeptide displaysimproved exo-specificity as shown in the table below.

Betamyl/ Variant t½-85 Phadebas Mutations pMD55 54 N33Y D34N G121F G134RA141P I157L L178F A179T G223A H307L S334P SSM219 B3 94 N26E N33Y D34NG121F G134R A141P I157L L178F A179T G223A H307L S334P

The half-life t1/2-85 is determined according to Example 8, aftergel-filtration of the samples with PD-10 columns (from AmershamBiosciences) using a 50 mM sodium citrate, 5 mM CaCl₂, pH 6.5 buffer.

Example 34 Enhanced Exo-Specificity of PS4 Variant Polypeptide withMutation 70D

A PS4 variant polypeptide designated SAS1401 L10 having amino acidmutations at N33Y D34N G70D G121F G134R A141P Y146G I157L G158T S161AL178F A179T G223E S229P H307L A309P S334P is tested for exo-specificity.This polypeptide displays improved exo-specificity as shown in the tablebelow.

Betamyl/ Variant t½-85 Phadebas Mutations pMD153 206 N33Y D34N G121FG134R A141P Y146G I157L G158T S161A L178F A179T G223E S229P H307L A309PS334P SAS1401 245 N33Y D34N G70D G121F G134R L10 A141P Y146G I157L G158TS161A L178F A179T G223E S229P H307L A309P S334P

The half-life t1/2-85 is determined according to Example 8, aftergel-filtration of the samples with PD-10 columns (from AmershamBiosciences) using a 50 mM sodium citrate, 5 mM CaCl₂, pH 6.5 buffer.

Example 35 Enhanced Thermostability and Exo-Specificity of PS4 VariantPolypeptide with Mutation 145D

A PS4 variant polypeptide designated SAS1387 D16 bf having amino acidmutations at N33Y D34N G121F G134R A141P N145D Y146G I157L G158T S161AL178F A179T G223E S229P H307L A309P S334P is tested for thermostabilityand exo-specificity. This polypeptide displays improved thermostabilityand improved exo-specificity as shown in the table below.

Betamyl/ Variant t½-85 Phadebas Mutations pMD153 12.3 206 N33Y D34NG121F G134R A141P Y146G I157L G158T S161A L178F A179T G223E S229P H307LA309P S334P SAS1387 22.3 336 N33Y D34N G121F G134R A141P D16 bf N145DY146G I157L G158T S161A L178F A179T G223E S229P H307L A309P S334P

The half-life t1/2-85 is determined according to Example 8, aftergel-filtration of the samples with PD-10 columns (from AmershamBiosciences) using a 50 mM sodium citrate, 5 mM CaCl₂, pH 6.5 buffer.

Example 36 Enhanced Thermostability of PS4 Variant Polypeptide withMutation 188H1

A PS4 variant polypeptide designated pMD236 having amino acid mutationsat N33Y D34N G70D G121F G134R A141P N145D Y146G I157L G158T S161A L178FA179T G188H G223E S229P H307L A309P S334P W339E is tested forthermostability. This polypeptide displays improved thermostability asshown in the table below.

Betamyl/ Variant t½-85 Phadebas Mutations pMD212 bf 12.2 N33Y D34N G70DG121F G134R A141P N145D Y146G I157L G158T S161A L178F A179T G223E S229PH307L A309P S334P W339E pMD236 16.1 N33Y D34N G70D G121F G134R A141PN145D Y146G I157L G158T S161A L178F A179T G188H G223E S229P H307L A309PS334P W339E

The half-life t1/2-85 is determined according to Example 8, aftergel-filtration of the samples with PD-10 columns (from AmershamBiosciences) using a 50 mM sodium citrate, 5 mM CaCl₂, pH 6.5 buffer.

Example 37 Enhanced Thermostability of PS4 Variant Polypeptide withMutation 188S

A PS4 variant polypeptide designated pMD237 bf having amino acidmutations at N33Y D34N G70D G121F G134R A141P N145D Y146G I157L G158TS161A L178F A179T G188S G223E S229P H307L A309P S334P W339E is testedfor thermostability. This polypeptide displays improved thermostabilityas shown in the table below.

Betamyl/ Variant t½-85 Phadebas Mutations pMD212 bf 12.2 N33Y D34N G70DG121F G134R A141P N145D Y146G I157L G158T S161A L178F A179T G223E S229PH307L A309P S334P W339E pMD237 bf 12.6 N33Y D34N G70D G121F G134R A141PN145D Y146G I157L G158T S161A L178F A179T G188S G223E S229P H307L A309PS334P W339E

The half-life t1/2-85 is determined according to Example 8, aftergel-filtration of the samples with PD-10 columns (from AmershamBiosciences) using a 50 mM sodium citrate, 5 mM CaCl₂, pH 6.5 buffer.

Example 38 Enhanced Exo-Specificity of PS4 Variant Polypeptide withMutation 339A

A PS4 variant polypeptide designated SAS1379 O13 having amino acidmutations at N33Y D34N G121F G134R A141P Y146G I157L G158T S161A L178FA179T G223E S229P H307L A309P S334P W339A is tested for exo-specificity.This polypeptide displays improved exo-specificity as shown in the tablebelow.

Betamyl/ Variant t½-85 Phadebas Mutations pMD153 206 N33Y D34N G121FG134R A141P Y146G I157L G158T S161A L178F A179T G223E S229P H307L A309PS334P SAS1379 301 N33Y D34N G121F G134R A141P O13 Y146G I157L G158TS161A L178F A179T G223E S229P H307L A309P S334P W339A

The half-life t1/2-85 is determined according to Example 8, aftergel-filtration of the samples with PD-10 columns (from AmershamBiosciences) using a 50 mM sodium citrate, 5 mM CaCl₂, pH 6.5 buffer.

Example 39 Enhanced Exo-Specificity of PS4 Variant Polypeptide withMutation 339E

A PS4 variant polypeptide designated SAS1379 O9 having amino acidmutations at N33Y D34N G121F G134R A141P Y146G I157L G158T S161A L178FA179T G223E S229P H307L A309P S334P W339E is tested for exo-specificity.This polypeptide displays improved exo-specificity as shown in the tablebelow.

Betamyl/ Variant t½-85 Phadebas Mutations pMD153 206 N33Y D34N G121FG134R A141P Y146G I157L G158T S161A L178F A179T G223E S229P H307L A309PS334P SAS1379 O9 347 N33Y D34N G121F G134R A141P Y146G I157L G158T S161AL178F A179T G223E S229P H307L A309P S334P W339E

The half-life t1/2-85 is determined according to Example 8, aftergel-filtration of the samples with PD-10 columns (from AmershamBiosciences) using a 50 mM sodium citrate, 5 mM CaCl₂, pH 6.5 buffer.

Further Aspects

Further aspects and embodiments of the invention are now set out in thefollowing numbered Paragraphs; it is to be understood that the inventionencompasses these aspects:

Paragraph A1. A PS4 variant polypeptide derivable from a parentpolypeptide having non-maltogenic exoamylase activity, in which the PS4variant polypeptide comprises an amino acid mutation at position 121with reference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1.

Paragraph A2. A PS4 variant polypeptide according to Paragraph A1, inwhich the mutation at position 121 comprises a substitution 121F, 121Yand/or 121W, preferably G121F, G121Y and/or G121W.

Paragraph A3. A PS4 variant polypeptide according to Paragraph A1 or A2,in which the PS4 variant polypeptide further comprises one or morefurther mutations at a position selected from the group consisting of:161 and 223.

Paragraph A4. A PS4 variant polypeptide according to Paragraph A3, inwhich the one or more further mutations is selected from the groupconsisting of: 161A, 223E and 223K, more preferably S161A, G223E and/orG223K.

Paragraph A5. A PS4 variant polypeptide according to any precedingParagraph A, in which the PS4 variant polypeptide comprises mutations atpositions selected from the group consisting of: 121, 161; 121, 223.

Paragraph A6. A PS4 variant polypeptide according to Paragraph A5, inwhich the PS4 variant polypeptide comprises mutations at positionsselected from the group consisting of: 121F/Y/W, 161A; 121F/Y/W, 223E/K.

Paragraph A7. A PS4 variant polypeptide according to any precedingParagraph A, in which the PS4 variant polypeptide comprises mutations atpositions selected from the group consisting of: 121, 161 and 223.

Paragraph A8. A PS4 variant polypeptide according to any precedingParagraph A, in which the PS4 variant polypeptide comprises mutations121F/Y/W, 161A, 223E/K.

Paragraph A9. A PS4 variant polypeptide according to any precedingParagraph A, in which the PS4 variant polypeptide further comprises oneor mutations, preferably all, selected from the group consisting ofpositions: 134, 141, 157, 223, 307, 334.

Paragraph A10. A PS4 variant polypeptide according to any precedingParagraph A, in which the PS4 variant polypeptide further comprisesmutations at either or both positions 33 and 34.

Paragraph A11. A PS4 variant polypeptide according to Paragraph A10, inwhich the PS4 variant polypeptide further comprises one orsubstitutions, preferably all, selected from the group consisting of:G134R, A141P, I157L, G223A, H307L, S334P, and optionally one or both ofN33Y and D34N.

Paragraph A12. A PS4 variant polypeptide according to any precedingParagraph A, in which the PS4 variant polypeptide further comprises:

(a) a mutation at position 121, preferably 121D, more preferably G121D;

(b) a mutation at position 178, preferably 178F, more preferably L178F;

(c) a mutation at position 179, preferably 179T, more preferably A179T;and/or

(d) a mutation at position 87, preferably 87S, more preferably G87S.

Paragraph B1. A PS4 variant polypeptide derivable from a parentpolypeptide having non-maltogenic exoamylase activity, in which the PS4variant polypeptide comprises an amino acid mutation at position 161with reference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1.

Paragraph B2. A PS4 variant polypeptide according to Paragraph B1, inwhich the mutation at position 161 comprises a substitution 161A,preferably S161A.

Paragraph B3. A PS4 variant polypeptide according to Paragraph B1 or B2,in which the PS4 variant polypeptide further comprises one or morefurther mutations at a position selected from the group consisting of:121 and 223.

Paragraph B4. A PS4 variant polypeptide according to Paragraph B3, inwhich the one or more further mutations is selected from the groupconsisting of: 121F, 121Y, 121W, 223E and 223K, more preferably G121F,G121Y, G121W, G223E and/or G223K.

Paragraph B5. A PS4 variant polypeptide according to any precedingParagraph B, in which the PS4 variant polypeptide comprises mutations atpositions selected from the group consisting of: 121, 161; 161, 223.

Paragraph B6. A PS4 variant polypeptide according to Paragraph B5, inwhich the PS4 variant polypeptide comprises mutations at positionsselected from the group consisting of: 121F/Y/W, 161A; 161A, 223E/K.

Paragraph B7. A PS4 variant polypeptide according to any precedingParagraph B, in which the PS4 variant polypeptide comprises mutations atpositions selected from the group consisting of: 121, 161 and 223.

Paragraph B8. A PS4 variant polypeptide according to any precedingParagraph B, in which the PS4 variant polypeptide comprises mutations121F/Y/W, 161A, 223E/K.

Paragraph B9. A PS4 variant polypeptide according to any precedingParagraph B, in which the PS4 variant polypeptide further comprises oneor mutations, preferably all, selected from the group consisting ofpositions: 134, 141, 157, 223, 307, 334.

Paragraph B110. A PS4 variant polypeptide according to any precedingParagraph B, in which the PS4 variant polypeptide further comprisesmutations at either or both positions 33 and 34.

Paragraph B131. A PS4 variant polypeptide according to Paragraph B110,in which the PS4 variant polypeptide further comprises one orsubstitutions, preferably all, selected from the group consisting of:G134R, A141P, I157L, G223A, H307L, S334P, and optionally one or both ofN33Y and D34N.

Paragraph B12. A PS4 variant polypeptide according to any precedingParagraph B, in which the PS4 variant polypeptide further comprises:

(a) a mutation at position 121, preferably 121D, more preferably G121D;

(b) a mutation at position 178, preferably 178F, more preferably L178F;

(c) a mutation at position 179, preferably 179T, more preferably A 79T;and/or

(d) a mutation at position 87, preferably 87S, more preferably G87S.

Paragraph C1. A PS4 variant polypeptide derivable from a parentpolypeptide having non-maltogenic exoamylase activity, in which the PS4variant polypeptide comprises an amino acid mutation at position 223with reference to the position numbering of a Pseudomonas saccharophiliaexoamylase sequence shown as SEQ ID NO: 1.

Paragraph C2. A PS4 variant polypeptide according to Paragraph C1, inwhich the mutation at position 223 comprises a substitution 223E and/or223K, preferably G223E and/or G223K.

Paragraph C3. A PS4 variant polypeptide according to Paragraph C1 or C2,in which the PS4 variant polypeptide further comprises one or morefurther mutations at a position selected from the group consisting of:121 and 161.

Paragraph C4. A PS4 variant polypeptide according to Paragraph C3, inwhich the one or more further mutations is selected from the groupconsisting of: 121F, 121Y, 121W and 161A, more preferably G121F, G121Y,G121W and/or S161A.

Paragraph C5. A PS4 variant polypeptide according to any precedingParagraph C, in which the PS4 variant polypeptide comprises mutations atpositions selected from the group consisting of: 121, 223; 161, 223.

Paragraph C6. A PS4 variant polypeptide according to Paragraph C5, inwhich the PS4 variant polypeptide comprises mutations at positionsselected from the group consisting of: 121F/Y/W, 223E/K; 161A, 223E/K.

Paragraph C7. A PS4 variant polypeptide according to any precedingParagraph C, in which the PS4 variant polypeptide comprises mutations atpositions selected from the group consisting of: 121, 161 and 223.

Paragraph C8. A PS4 variant polypeptide according to any precedingParagraph C, in which the PS4 variant polypeptide comprises mutations121F/Y/W, 161A, 223E/K.

Paragraph C9. A PS4 variant polypeptide according to any precedingParagraph C, in which the PS4 variant polypeptide further comprises oneor mutations, preferably all, selected from the group consisting ofpositions: 134, 141, 157, 223, 307, 334.

Paragraph C10. A PS4 variant polypeptide according to any precedingParagraph C, in which the PS4 variant polypeptide further comprisesmutations at either or both positions 33 and 34.

Paragraph C11. A PS4 variant polypeptide according to Paragraph C10, inwhich the PS4 variant polypeptide further comprises one orsubstitutions, preferably all, selected from the group consisting of:G134R, A141P, I157L, G223A, H307L, S334P, and optionally one or both ofN33Y and D34N.

Paragraph C12. A PS4 variant polypeptide according to any precedingParagraph C, in which the PS4 variant polypeptide further comprises:

(a) a mutation at position 121, preferably 121D, more preferably G121D;

(b) a mutation at position 178, preferably 178F, more preferably L178F;

(c) a mutation at position 179, preferably 179T, more preferably A179T;and/or

(d) a mutation at position 87, preferably 87S, more preferably G87S.

Paragraph D1. A PS4 variant polypeptide derivable from a parentpolypeptide having non-maltogenic exoamylase activity, in which the PS4variant polypeptide comprises an amino acid mutation at one or morepositions selected from the group consisting of: 146, 157, 158, 198,229, 303, 306, 309, 316 and 353, with reference to the positionnumbering of a Pseudomonas saccharophilia exoamylase sequence shown asSEQ ID NO: 1.

Paragraph D2. A PS4 variant polypeptide according to Paragraph D1, inwhich the PS4 variant polypeptide comprises an amino acid mutationselected from the group consisting of: 146G, 146M, 157M, 158T, 158A,158S, 198W, 198F, 229P, 303E, 303D, 306T, 306G, 309P, 316S, 316P, 316K,316Q and 353T.

Paragraph D3. A PS4 variant polypeptide according to Paragraph D1, inwhich the PS4 variant polypeptide comprises an amino acid mutation 146G,157M, 158T, 198W, 229P, 303E, 303D, 306T, 306G, 309P, 316S, 316P or353T.

Paragraph D 4. A PS4 variant polypeptide according to Paragraph D1, inwhich the PS4 variant polypeptide further comprises one or moremutations selected from the group consisting of positions: 33, 34, 121,134, 141, 157, 161, 178, 179, 223, 307 and 334, preferably selected fromthe group consisting of: 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F,179T, 223E, 307L and 334P.

Paragraph D5. A PS4 variant polypeptide according to Paragraph D1, inwhich the PS4 variant polypeptide comprises each of the followingmutations:

(a) 33Y, 34N, 121F, 134R, 141P, 146G, 157L, 161A, 178F, 179T, 223E, 307Land 334P, preferably having a sequence SEQ ID NO: 15;

(b) 33Y, 34N, 121F, 134%, 141P, 157, 161A, 178F, 179T, 223E, 307L and334P, preferably having a sequence SEQ ID NO: 16;

(c) 33Y, 34N, 121F, 134R, 141P, 157L, 158T, 161A, 178F, 179T, 223E, 307Land 334P, preferably having a sequence SEQ ID NO: 17;

(d) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 198W, 223E, 307Land 334P, preferably having a sequence SEQ ID NO: 18;

(e) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 229P, 307Land 334P, preferably having a sequence SEQ ID NO: 19;

(f) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 198W, 223E,229P, 307L and 334P, preferably having a sequence SEQ ID NO: 20;

(g) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 303E, 307Land 334P, preferably having a sequence SEQ ID NO: 21;

(h) 33Y, 34N, 121F, 134R, 141P, 157, 161A, 178F, 179T, 223E, 303D, 307Land 334P, preferably having a sequence SEQ ID NO: 22;

(i) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 306T, 307Land 334P, preferably having a sequence SEQ ID NO: 23;

(j) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 306G, 307Land 334P, preferably having a sequence SEQ ID NO: 24;

(k) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 309P, 307Land 334P, preferably having a sequence SEQ ID NO: 25;

(l) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 307L,316S, and 334P, preferably having a sequence SEQ ID NO: 26;

(m) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 307L, 316Pand 334P, preferably having a sequence SEQ ID NO: 27; and,

(n) 33Y, 34N, 121F, 134R, 141P, 1571, 161A, 178F, 179T, 223E, 307L, 334Pand 353T, preferably having a sequence SEQ ID NO: 28.

Paragraph 13. A PS4 variant polypeptide according to any precedingParagraph, in which the parent polypeptide comprises a non-maltogenicexoamylase, preferably a glucan 1,4-alpha-maltotetrahydrolase (EC3.2.1.60).

Paragraph 14. A PS4 variant polypeptide according to any precedingParagraph, in which the parent polypeptide is or is derivable fromPseudomonas species, preferably Pseudomonas saccharophilia orPseudomonas stutzeri.

Paragraph 15. A PS4 variant polypeptide according to any precedingParagraph, in which the parent polypeptide is a non-maltogenicexoamylase from Pseudomonas saccharophilia exoamylase having a sequenceshown as SEQ ID NO: 1 or SEQ ID NO: 5.

Paragraph 16. A PS4 variant polypeptide according to any of ParagraphsA1 to A12, B1 to B12, C1 to C12, D1 to D5, 13 and 14, in which theparent polypeptide is a non-maltogenic exoamylase from Pseudomonasstutzeri having a sequence shown as SEQ ID NO: 7 or SEQ ID NO: 11.

Paragraph 17. A PS4 variant polypeptide according to any precedingParagraph, which comprises a sequence as set out in the description orParagraphs.

Paragraph 18. A PS4 variant polypeptide according to any precedingParagraph, in which the PS4 variant polypeptide has a higherthermostability compared to the parent polypeptide or a wild typepolypeptide when tested under the same conditions.

Paragraph 19. A PS4 variant polypeptide according to any precedingParagraph, in which the half life (t1/2), preferably at 60 degrees C.,is increased by 15% or more, preferably 50% or more, most preferably100% or more, relative to the parent polypeptide or the wild typepolypeptide.

Paragraph 20. A PS4 variant polypeptide according to any precedingParagraph, in which the PS4 variant polypeptide has a higherexo-specificity compared to the parent polypeptide or a wild typepolypeptide when tested under the same conditions.

Paragraph 21. A PS4 variant polypeptide according to any precedingParagraph, in which the PS4 variant polypeptide has 10% or more,preferably 20% or more, preferably 50% or more, exo-specificity comparedto the parent polypeptide or the wild type polypeptide.

Paragraph 22. Use of a PS4 variant polypeptide as set out in anypreceding Paragraph as a food additive.

Paragraph 23. A process for treating a starch comprising contacting thestarch with a PS4 variant polypeptide as set out in any of Paragraphs A1to A12, B1 to B12, C1 to C12, D1 to D5 and 13 to 21 and allowing thepolypeptide to generate from the starch one or more linear products.

Paragraph 24. Use of a PS4 variant polypeptide as set out in any ofParagraphs A1 to A12, B1 to B12, C1 to C12, D1 to D5 and 13 to 21 inpreparing a food product.

Paragraph 25. A process of preparing a food product comprising admixinga polypeptide as set out in any of Paragraphs A1 to A12, B1 to B12, C1to C12, D1 to D5 and 13 to 21 with a food ingredient.

Paragraph 26. Use according to Paragraph 24, or a process according toParagraph 25, in which the food product comprises a dough or a doughproduct, preferably a processed dough product.

Paragraph 27. A use or process according to any of Paragraphs 24 to 26,in which the food product is a bakery product.

Paragraph 28. A process for making a bakery product comprising: (a)providing a starch medium; (b) adding to the starch medium a PS4 variantpolypeptide as set out in any of Paragraphs A1 to A12, B1 to B12, C1 toC12, D1 to D5 and 13 to 21; and (c) applying heat to the starch mediumduring or after step ad) to produce a bakery product.

Paragraph 29. A food product, dough product or a bakery product obtainedby a process according to any of Paragraphs 24 to 28.

Paragraph 30. An improver composition for a dough, in which the improvercomposition comprises a PS4 variant polypeptide as set out in any ofParagraphs A1 to A12, B1 to B12, C1 to C12, D1 to D5 and 13 to 21, andat least one further dough ingredient or dough additive.

Paragraph 31. A composition comprising a flour and a PS4 variantpolypeptide as set out in any of Paragraphs 1 to 21.

Paragraph 32. Use of a PS4 variant polypeptide as set out in any ofParagraphs A1 to A12, B1 to B12, C1 to C12, D1 to D5 and 13 to 21, in adough product to retard or reduce staling, preferably detrimentalretrogradation, of the dough product.

Paragraph 33. A combination of a PS4 variant polypeptide as set out inany preceding Paragraph, together with Novamyl, or a variant, homologue,or mutants thereof which has maltogenic alpha-amylase activity.

Paragraph 34. Use of a combination according to Paragraph 33 for anapplication according to any preceding Paragraph.

Paragraph 35. A food product produced by treatment with a combinationaccording to Paragraph 34.

Paragraph 36. A food additive comprising a PS4 variant polypeptideaccording to any of Paragraphs A1 to A12, B1 to B12, C1 to C12, D1 to D5and 13 to 21.

REFERENCES

-   Penning a, D., van der Veen, B. A., Knegtel, R. M., van Hijum, S.    A., Rozeboom, H. J., Kan K. H., Dijkstra, B. W., Dijkhuizen, L.    (1996). The raw starch binding domain of cyclodextrin    glycosyltransferase from Bacillus circulans strain 251. J. Biol.    Chem. 271, 32777-32784.-   Sambrook J, F. E. M. T. (1989). Molecular Cloning: A Laboratory    Manual, 2nd Edn. Cold Spring Harbor Laboratory, Cold Spring Harbor    N.Y.-   Zhou, J. H., Baba, T., Takano, T., Kobayashi, S., Arai, Y. (1989).    Nucleotide sequence of the maltotetraohydrolase gene from    Pseudomonas saccharophila FEBS Lett. 255, 37-41.

Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for caring out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

The invention claimed is:
 1. A non-naturally occurring variantpolypeptide of SEQ ID NO: 1 having non-maltogenic exoamylase activityand comprising an amino acid mutation at position 146 with reference tothe position numbering of SEQ ID NO:
 1. 2. The polypeptide according toclaim 1, wherein the polypeptide comprises an amino acid mutationselected from the group consisting of 121F, 121Y, 121W, 161A, 223E,223K, 146G, 146M, 157M, 158T, 158A, 158S, 198W, 198F, 229P, 303E, 303D,306T, 306G, 309P, 316S, 316P, 316K, 316Q, 353T, 26E, 70D, 145D, 188S,188T, 188H, 272Q, 339A and 339E.
 3. The polypeptide according to claim1, wherein the polypeptide further comprises one or more mutationsselected from the group consisting of positions: 33, 34, 121, 134, 141,157, 161, 178, 179, 223, 307 and
 334. 4. The polypeptide according toclaim 1, wherein the polypeptide comprises each of the followingmutations selected from a group consisting of: (a) 33Y, 34N, 121F, 134R,141P, 146G, 157L, 161A, 178F, 179T, 223E, 307L and 334P; (b) 33Y, 34N,121F, 134R, 141P, 157M, 161A, 178F, 179T, 223E, 307L and 334P; (c) 33Y,34N, 121F, 134R, 141P, 157L, 158T, 161A, 178F, 179T, 223E, 307L and334P; (d) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 198W,223E, 307L and 334P; (e) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F,179T, 223E, 229P, 307L and 334P; (f) 33Y, 34N, 121F, 134R, 141P, 157L,161A, 178F, 179T, 198W, 223E, 229P, 307L and 334P; (g) 33Y, 34N, 121F,134R, 141P, 157L, 161A, 178F, 179T, 223E, 303E, 307L and 334P; (h) 33Y,34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 303D, 307L and334P; (i) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,306T, 307L and 334P; j) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F,179T, 223E, 306G, 307L and 334P; (k) 33Y, 34N, 121F, 134R, 141P, 157L,161A, 178F, 179T, 223E, 309P, 307L and 334P; (l) 33Y, 34N, 121F, 134R,141P, 157L, 161A, 178F, 179T, 223E, 307L, 316S, and 334P; (m) 33Y, 34N,121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 307L, 316P and 334P; (n)33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E, 307L, 334P and353T; and (o) 33Y, 34N, 121F, 134R, 141P, 157L, 161A, 178F, 179T, 223E,307L and 334P.
 5. The polypeptide according to claim 1 wherein thepolypeptide comprises each of the following mutations selected from agroup consisting of: (a) N26E, N33Y, D34N, G121F, G134R, A141P, I157L,L178F, A179T, G223A, H307L, S334P; (b) N33Y, D34N, G70D, G121F, G134R,A141P, Y146G, I157L, G158T, S161A, L178F, A179T, G223E, S229P, H307L,A309P, S334P; (c) N33Y, D34N, G121F, G134R, A141P, N145D, Y146G, I157L,G158T, S161A, L178F, A179T, G223E, S229P, H307L, A309P, S334P; (d) N33Y,D34N, G70D, G121F, G134R, A141P, N145D, Y146G, I157L, G158T, S161A,L178F, A179T, G188H, G223E, S229P, H307L, A309P, S334P, W339E; (e) N33Y,D34N, G70D, G121F, G134R, A141P, N145D, Y146G, I157L, G158T, S161A,L178F, A179T, G188S, G223E, S229P, H307L, A309P, S334P, W339E; (f) N33Y,D34N, G121F, G134R, A141P, Y146G, I157L, G158T, S161A, L178F, A179T,G223E, S229P, H307L, A309P, S334P, W339A; and (g) N33Y, D34N, G121F,G134R, A141P, Y146G, I157L, G158T, S161A, L178F, A179T, G223E, S229P,H307L, A309P, S334P, W339E.
 6. The polypeptide according to claim 1,wherein the polypeptide further comprises a mutation at position 87,wherein said mutation is G87S.
 7. The polypeptide according to claim 1,wherein the polypeptide has a higher thermostability compared to SEQ IDNO: 1 when tested under the same conditions.
 8. The polypeptideaccording to claim 7, in which half life (t1/2) at 60 degrees C., isincreased by 15% or more relative to SEQ ID NO:
 1. 9. The polypeptideaccording to claim 1, wherein the polypeptide has a higherexo-specificity compared to SEQ ID NO: 1 when tested under the sameconditions.
 10. The polypeptide according to claim 9, wherein thepolypeptide has 10% or more exo-specificity relative to SEQ ID NO: 1 .11. A process of preparing a food or feed product comprising admixingsaid polypeptide of claim 1 with a food or feed ingredient.
 12. Aprocess for making a bakery product comprising: (a) providing a starchmedium; (b) adding to the starch medium said polypeptide of claim 1; and(c) applying heat to the starch medium during or after step (b) toproduce a bakery product.
 13. A food product, feed product, doughproduct or a bakery product obtained by a process according to claim 12.14. An improver composition for a dough, in which the improvercomposition comprises a polypeptide as set out in claim 1, and at leastone further dough ingredient or dough additive.
 15. A compositioncomprising a flour and the polypeptide as set out in claim
 1. 16. Acombination of the variant polypeptide as set out in claim 1, togetherwith an enzyme having maltogenic alpha-amylase activity.
 17. A food orfeed product produced by treatment with a combination according to claim16.
 18. A process according to claim 11, in which the food productcomprises a dough or a dough product, preferably a processed doughproduct.
 19. A process according to claim 11, in which the food productis a bakery product.
 20. The polypeptide according to claim 1,comprising a further amino acid mutation at one or more positionsselected from the group consisting of 158, 198, 229, 303, 306, 309, 316,353, 26, 70, 145, 188, 272 and 339.